Compositions and methods for treating vascular disorders

ABSTRACT

The present disclosure provides compositions and methods of their use to impair angiogenesis in a patient to treat indications including, but not limited to, tumor growth, age-related macular degeneration, and metastasis. Also provided are compositions and methods for promoting angiogenesis in patients in need thereof.

PRIORITY CLAIM

This application claims benefit of priority to U.S. ProvisionalApplication Ser. No. 62/304,015, filed Mar. 4, 2016, the entire contentsof which are hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with U.S. government support under NationalInstitutes of Health Grant Nos. EY021862 and EY026069. The U.S.government has certain rights in this invention.

BACKGROUND

Vascular endothelial cells (ECs) line the internal surface of all bloodvessels and are essential for vascular development and homeostasis. Thede novo generation of blood vessels, known as vasculogenesis, is drivenby the proliferation, migration and networking of endothelial progenitorcells. Subsequently, sprouting angiogenesis and intussusceptiveangiogenesis drive new blood vessel formation from existing vessels,which is responsible for the expansion of vascular system. In the adult,the endothelium is normally quiescent, but angiogenesis can occur inresponse to stimuli. Physiological angiogenesis is associated with woundhealing, post-ischemic tissue restoration and the menstrual cycle, whilepathological angiogenesis is associated with numerous diseases,including cancer, age-related macular degeneration (AMD), diabeticretinopathy and atherosclerosis. Major breakthroughs have been made inthe last decades regarding the cellular and molecular mechanism ofangiogenesis. Vascular endothelial growth factor (VEGF) has beenestablished as a major growth factor for ECs. Anti-angiogenic therapy,such as anti-VEGF antibodies, has shown efficacy in treating wet AMD andsome cancers. However, in many cases the efficacy of anti-angiogenictherapy is still limited, suggesting complex mechanisms of angiogenesis.This necessitates a thorough mechanistic investigation of angiogenesisand the development of novel and alternative therapeutic approaches.

It is now established that up to 90% of the human genome is transcribed,and the majority of these transcripts are non-coding RNAs (ncRNAs) thatdo not encode proteins. ncRNAs can be classified as short noncoding RNAssuch as microRNAs (miRNAs), long noncoding RNAs (lncRNAs) and otherclassic ncRNAs. miRNAs include a group of small noncoding RNAs sized ˜22nucleotides that play important regulatory functions in numerousphysiological and pathological processes, including angiogenesis.lncRNAs represent a large group of long (typically >200 nt) noncodingRNAs, whose function is still largely enigmatic. A small number ofwell-studied lncRNAs have given us important clues about theirbiological function. lncRNAs have been shown to play key roles indiverse processes including genomic imprinting, cell cycle regulationand cell identity determination. Dependent on their subcellularlocalizations, lncRNAs may play a cis-acting regulatory function onnearby genes, regulate chromatin-modification through interacting withother factors, or regulate protein translation or miRNA function in thecytoplasm.

Several lncRNAs have been implicated in cardiovascular biology. Thelateral plate mesoderm-specific lncRNA Fendrr has been shown to berequired for proper heart and body wall development, and the lncRNABraveheart (Bvht) is essential for cardiomyocyte lineage commitment. Arecent study showed that overexpression of a cardiac-5 specificlncRNAMhrt was able to protect against pathological hypertrophy in mice.The expression, regulation and function of lncRNAs in vasculature islargely unknown. A recent characterization of lncRNAs in human umbilicalvein ECs (HUVECs) identified an lncRNA named metastasis-associated lungadenocarcinoma transcript 1 (MALAT1) that is highly expressed in HUVECs.MALAT1 functions to tip the balance from EC proliferation to migrationand is required for vessel growth in vivo. RNA sequencing of humancoronary smooth muscle cells (SMCs) has identified 31 unannotatedlncRNAs, one of which is a vascular SMC- and EC-enriched lncRNA calledSENCR, which functions to inhibit the migration of VSMCs. AnotherlncRNA, lncRNA-p21, functions to repress proliferation and induceapoptosis of VSMCs in vitro and in vivo, and is downregulated inatherosclerotic plaques of ApoE−/− mice and coronary artery tissues ofhuman coronary artery disease patients. Overall, the role of lncRNAs inangiogenesis, especially human angiogenesis, is largely unknown.

SUMMARY

In one embodiment the present disclosure provides compositions comprisedof small interfering ribonucleic acids (siRNAs) and methods of usethereof to silence lncEGFL7OS and repress angiogenesis in humans. Inanother embodiment the present disclosure provides compositionscomprised of viruses overexpressing lncEGFL7OS and methods of usethereof to activate lncEGFL7OS and promote angiogenesis in humans. Inanother embodiment, overexpression or silencing of the genes thatregulate lncEGFL7OS expression to promote or inhibit angiogenesis alsoare contemplated. Such genes include ETS1, ETS2 and MAX.

In another embodiment, there is provided a composition comprising anagonist or antagonist of lncEGFL7OS function in a pharmaceuticallyacceptable buffer, diluent or medium. The antagonist may comprise one ormore of si-LncEGFL7OS#1 and si-LncEGFL7OS#2, or an expression vectorcoding for the same. The agonist may be lncEGFL7OS or an expressionvector coding for the same. The expression vector may be a viral vectoror a non-viral vector. The agonist or antagonist may be a nucleic acidanalog of lncEGFL7OS or that hybridizes to lncEGFL7OS. The nucleic acidanalog may comprise one or more non-natural bases. The composition mayfurther comprise an agonist or antagonist of miR-126.

In yet another embodiment, there is provided a method of promotingvascular integrity and/or vascular repair comprising administering to asubject at risk of or suffering from vascular damage an agonist oflncEGFL7OS function. The subject may be suffering from vascular damage,such as a cardiac tissue, and/or from an ischemic event. The ischemicevent may comprise an infarct, ischemia-reperfusion injury or arterialstenosis. The vascular damage may be directed to a non-cardiac tissue,such as from trauma or vascular leakage. The subject may be at risk ofvascular damage, such as due to hypertension, late stage atherosclerosiscardiac hypetrophy, osteoporosis, neurodegeneration, fibrosis orrespiratory distress. The subject may be a non-human animal or a human.

The agonist may be lncEGFL7OS or a mimetic of lncEGFL7OS. The agonistmay be an expression vector comprising an lncEGFL7-encoding nucleic acidsegment under the control of a promoter active in a target cell. Thetarget cell may be an endothelial cell or a hematopoietic cell. Thepromoter may be a tissue selective/specific promoter, such as one activein an endothelial cell or a hematopoietic cell. The expression vectormay be a viral vector or a non-viral vector.

The method may further comprise administering to said subject asecondary therapy. The administering comprises systemic administration,such as oral, intravenous, or intra-arterial. Administering may be byosmotic pump or catheter. Administration may be directly to or local tovascular damaged tissue or a tissue at risk of vascular damage, such asto cardiac tissue, blood vessel tissue, bone tissue, neuronal tissue,respiratory tissue, eye tissue or placental tissue.

In still a further embodiment, there is provided a method of inhibitingpathologic vascularization in a subject in need thereof comprisingadministering to the subject at risk of or suffering from pathologicvascularization an antagonist of lncEGFL7OS. The subject may besuffering from pathologic vascularization, such as that associated withearly stage atherosclerosis, retinopathy, cancer, age-related maculardegeneration or stroke. The subject may be at risk of pathologicvascularization, such as that associated with hyperlipidemia, obesity,asthma, arthritis, psoriasis and/or blindness. The subject may be anon-human animal or a human.

The antagonist maybe a lncEGFL7OS antisense molecule, or an inhibitor ofETS1, ETS2 and/or MAX expression or function. The antagonist may bedelivered to a vasculature tissue, smooth muscle, ocular tissue,hematopoietic tissue, bone marrow, lung tissue or an epicardial tissue.The method may further comprise administering to said subject asecondary anti-angiogenic therapy. Administering may comprise systemicadministration, such as oral, intravenous, intra-arterialadministration. Administration may be directly to or local to pathologicvascularization or a tissue at risk of pathologic vascularization, suchas to ocular tissue, a vascular tissue, bone tissue, fat tissue or lungtissue. Administering may be by osmotic pump or catheter.

In still yet an additional embodiment, the present disclosure providescompositions comprised of small interfering ribonucleic acids (siRNAs)and methods of use thereof to activate or silence the followingEC-enriched lncRNAs to repress or activate angiogenesis in humans:Friend leukemia integration 1 (FLI1) antisense lncRNA (lncFLI1,ASHGA5P026051 (also known as SENCR), GATA binding protein 2 (GATA2)antisense lncRNA (lncGATA2, ASHGA5P019223, RP11-475N22.4), endothelialconverting enzyme 1 (ECE1) intron sense-overlapping lncRNA (lncECE1,ASHGA5P032664, AX747766), endothelial cell-selective adhesion molecule(ESAM) bidirectional lncRNA (lncESAM, ASHGA5P021448, RP11-677M14.3),roundabout homolog 4 (ROBO4) nature antisense RNA (lncROBO4,ASHGA5P026882, RP11-664121.5).

While certain features of this disclosure shown and described below arepointed out in the annexed claims, the disclosure is not intended to belimited to the details specified, since a person of ordinary skill inthe relevant art will understand that various omissions, modifications,substitutions and changes in the forms and details of the disclosureillustrated and in its operation may be made without departing in anyway from the spirit of the present disclosure. No feature of thedisclosure is critical or essential unless it is expressly stated asbeing “critical” or “essential.”

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure. The disclosure may be better understood by reference to oneor more of these drawings in combination with the description ofspecific embodiments presented herein.

FIGS. 1A-D: FIG. 1A. Hierarchy cluster analysis of lncRNA and mRNAexpression data from 5 different cell lines. FIG. 1B. Heatmap showingthe top-50 enriched lncRNAs in three EC lines compared to the two non-EClines. FIG. 1C. A pie chart showing different classes of annotatedlncRNAs that are enriched more than 2-fold in ECs compared to non-ECs.FIG. 1D. Functional enrichment analysis of EC-enriched lncRNAs and theirassociated genes.

FIGS. 2A-B: FIG. 2A. Quantitative RT-PCR confirmation of candidateEC-enriched lncRNAs. FIG. 2B. Tissue distribution of the candidatelncRNAs from the top-50 enriched lncRNA list.

FIGS. 3A-G: Expression and subcellular localization of lncEGFLOS. FIG.3A. Quantitative (q) RT-PCR confirmation of candidate EC-enrichedlncRNAs. FIG. 3B. Genomic organization of lncEGFL7OS and its host geneEGFL7/miR-126. Exons are shown in orange and the introns are shown inblue. Direction of gene transcription is indicated by arrows. Scale=1kb. FIG. 3C. Relative lncEGFL7OS expression level in a human total RNAmaster panel II (clontech). GAPDH served as the normalization control.FIG. 3D. Expression of EGFL7B and EGFL7C by qRT-PCR in different humantissues. GAPDH served as the normalization control. FIG. 3E. RelativemiR-126 expression level in different human tissues. U6 served asnormalization control. FIG. 3F. Expression of lncEGFL7OS in the nucleusand cytoplasm of HUVECs shown by semi-quantitative RT-PCR. RT-PCRshowing nuclear and cytoplasmic expression of lncEGFL7OS. SENCR was useda marker for cytoplasmic-enriched lncRNA, while NEAT-1 was used as amarker for nuclear-enriched lncRNA. FIG. 3G. Expression of lncEGFL7OS inthe nucleus and cytoplasm of HUVECs shown by high-resolution RNA FISHanalysis (a-b). High resolution RNA FISH experiments using lncEGFL7OS,NEAT1 and PP1B probes. PP1B was used as a marker for cytoplasmic mRNAwhile NEAT-1 was used as a marker for nuclear enriched lncRNA. Scale Barequals 5 μm.

FIGS. 4A-F: FIG. 4A. Impaired EC tube formation after lncEGFL7OSsilencing by in vitro Matrigel assay. Scale bar equals to 100 FIG. 4B.Quantification of branching points in the Matrigel assay in FIG. 4A.***, p<0.001. FIG. 4C. Decreased capillary tube formation at 7 and 9days after lncEGFL7OS silencing in an EC-fibroblast co-culture assay.The capillaries are stained with vWF antibody. Scale bar equals to 250FIG. 4D. Quantification of vessel density in FIG. 4C. *, p<0.05; ***,p<0.001. FIG. 4E. Defective EC networking at 14 days after lncEGFL7OSsilencing in an in vivo Matrigel implantation model. FIG. 4F.Quantification of tubule length in FIG. 4E. *, p<0.001.

FIGS. 5A-E: FIG. 5A. Quantification of EC proliferation in response toVEGF-A as indicated by BrDU 10 incorporation after lncEGFL7OS silencing.FIG. 5B. Quantification of cell cycle profile in ECs after lncEGFL7OSsilencing. FIG. 5C. Repression of cell migration in a scratch woundassay in ECs after lncEGFL7OS silencing. Dashed lines indicate theinitial position of cells. Scale bar equals to 100 FIG. 5D.Quantification of EC migration in C. **, p<0.01. FIG. 5E. Quantificationof TUNEL positive cells in ECs transfected siRNAs for lncEGFL7. *,p<0.05.

FIGS. 6A-H: Regulation of EGFL7/miR-126 and angiogenic signaling bylncEGFL7OS. FIG. 6A. Expression of EGFL7 isoforms by qRT-PCR afterlncEGFL7OS knockdown in ECs (n=3). GAPDH served as normalizationcontrol. FIG. 6B. Expression of EGFL7 protein by Western blot afterlncEGFL7OS knockdown in ECs (n=3). β-Tubulin was used as a loadingcontrol. FIG. 6C. Expression of miR-126 and miR-126* after lncEGFL7OSknockdown in ECs (n=3). U6 or miR-24 served as normalization control.FIG. 6D. Regulation of ERK1/2 and AKT phosphorylation by lncEGFL7OSknockdown in ECs in response to VEGF treatment, as revealed by Westernblot. Total ERK1/2 and AKT were used as controls. β-Tubulin was used asa loading control. FIG. 6E. qRT-PCR showing upregulation of miR-126expression in ECs infected with lncEGFL7OS expressing adenovirus. GFPexpression virus was used as control. ***, p<0.001. FIG. 6F. qRT-PCRshowing upregulation of EGFL7B expression in ECs infected withlncEGFL7OS expressing adenovirus. GFP expression virus was used ascontrol. ***, p<0.001. FIG. 6G. Partial rescue of thelncEGFL7OS-knockdown angiogenic phenotype by miR-126 mimic in an ECfibroblast co-culture assay. Scale bar equals to 400 μm. FIG. 6H.Quantification of vessel area in G (n=3). **, P<0.01. ***, p<0.001.

FIGS. 7A-D: FIG. 7A. Inhibition of lncEGFL7OS expression bysi-lncEGFL7-1/2 in human choroids cultured ex vivo, as revealed byreal-time RT-PCR. FIG. 7B. Representative picture of human choroidsprouting angiogenesis after lncEGFL7OS knockdown. FIG. 7C.Quantification of choroid sprouting distance in FIG. 7B. FIG. 7D. ICAM2(green) and Isolectin B4 (red) staining of the choroid sprouts in FIG.7B. Scale bar equals to 250 μm.

FIGS. 8A-B: FIG. 8A. Uptake of DiI-labeled Acetyl-LDL in the EC lines.FIG. 8B. Staining of the EC lines with antibody to EC marker vWF.

FIG. 9: Expression of EGFL7 isoforms by RT-PCR in different humantissues. β-Tubulin was used as a loading control.

FIG. 10: Silencing of lncEGFL7OS by siRNAs as shown by real-time RT-PCR.

FIG. 11: List of lncRNAs that are enriched more than 2-fold in three EClines (HUVEC, HREC and HCEC) than in two non-EC lines (HDF and ARPE-19).

FIGS. 12A-G: FIG. 12A. Quantitative RT-PCR confirmation of candidateEC-enriched lncRNAs. FIG. 12B. Genomic organization of lncEGFL7OS andits host 10 gene EGFL7/miR-126. Exons are shown in orange and theintrons are shown in blue. Direction of gene transcription is indicatedby arrows. Scale=1 kB. FIG. 12C. Relative lncEGFL7OS expression level indifferent human tissues. GAPDH served as the normalization control. FIG.12D. Expression of EGFL7 isoforms by RT-PCR in different human tissues.β-Tubulin was used as a loading control. FIG. 12E. Relative miR-126expression level in different human tissues. U6 served as normalizationcontrol. FIG. 12F. Expression of lncEGFL7OS in the nucleus and cytoplasmof HUVECs shown by semi-quantitative RT-PCR. RT-PCR showing nuclear andcytoplasmic expression of lncEGFL7OS. SENCR was used a marker forcytoplasmic-enriched lncRNA, while NEAT-1 was used as a marker fornuclear-enriched-lncRNA. FIG. 12G. Expression of lncEGFL7OS in thenucleus and cytoplasm of HUVECs shown by high-resolution RNA FISHanalysis (a-b). High resolution RNA FISH experiments using lncEGFL7OS,NEAT1 and PP1A probes. PP1A was used as a marker for cytoplasmic mRNAwhile NEAT-1 was used as a marker for nuclear-enriched lncRNA. Scale Barequals 5 μm.

FIGS. 13A-D: FIG. 13A. Schematic potential promoter region (boxed) forEGFL7/lncEGFL7OS. This region was shown by ENCODE to bind MAX, MYC,ETS1, RNA PolR II, H3K4Me1 and H3K27Ac. FIG. 13B. ChIP assay showingbinding for the indicated factors to the promoter region (region 2). IGGwas used as control. Primers for PCRs were shown in FIG. 13A. FIG. 13C.Testing bidirectional lncEGFL7OS promoter. LncEGF7OS promoter was fusedto a promoter-less Luciferase vector in forward (F) and reverse (R)directions, and tested for Luciferase activity with or withoutco-transfection of increasing amount of ETS1 or ETS1 mutant expressionplasmid in 293T cells. FIG. 13D. qRT-PCR showing that silencing ofETS1/2 result in the downregulation of lncEGFL7OS and primiR-126. *,p<0.05; ***, p<0.001.

FIGS. 14A-C: Overexpression of lncEGFL7OS enhances angiogenesis in anEC/Fibroblast co-culture assay. FIG. 14A. qRT-PCR showing overexpressionof lncEGFL7OS in ECs infected with lncEGFL7OS expressing adenovirus. GFPexpression virus was used as control.

FIG. 14B. Enhanced angiogenesis at 9 days after lncEGFL7OSoverexpression in an EC-fibroblast co-culture assay. Scale bar equals to500 FIG. 14C. Quantification of tube length/area in FIG. 19B (n=3). **,P<0.01.

FIGS. 15A-J: lncEGFL7OS regulates EGFL7/miR-126 transcription byinteraction with MAX transcription factor and enhancement of H3K27acetylation. FIG. 15A. Schematic EGFL7/miR-126 enhancer/promoter region.The boxed region is predicted by ENCODE to bind MAX and H3K27Ac. FIG.15B. ChIP-PCR showing specific binding of MAX to region 3 in FIG. 15A.Overexpression of lncEGFL7OS enhances MAX binding to the region. *,p<0.05; **, p<0.01. FIG. 15C. ChIP-PCR showing specific binding ofH3K27ac to region 3 in FIG. 15A. Overexpression 917 of lncEGFL7OSenhances MAX binding to the region. *, p<0.05; ***, p<0.001. FIG. 15D.RIP-PCR showing binding of MAX to lncEGFL7OS in ECs. Overexpression oflncEGFL7OS by adenovirus enhances MAX binding. The bottom line shows anon-RT control for PCR. FIG. 15E. Silencing of MAX expression by twoindependent siRNAs as shown by qRT-PCR. ***, p<0.001. FIG. 15F.Downregulation of EGFL7B by MAX silencing in ECs. **, p<0.01, ***,p<0.001. FIG. 15G. Downregulation of lncEGFL7OS by MAX silencing in ECs.**, p<0.01, ***, p<0.001. FIG. 15H. Downregulation of miR-126 by MAXsilencing in ECs. **, p<0.01. FIG. 15I. Quantification of vessel densityin an EC-Fibroblast co-culture assay after MAX silencing. A mix of twoindependent MAX siRNAs was used in the assay. **, p<0.01. FIG. 15J. MAXsilencing blunts the induction of miR-126 by lncEGFL7OS-expressingadenovirus. ***, p<0.001.

FIG. 16: A model for lncEGFL7OS in human angiogenesis. LncEGFL7OS istranscribed in the opposite strand of EGFL7/miR-126 gene under thecontrol of an ETS transcription factors-regulated bidirectionalpromoter. In turn, lncEGFL7OS transcripts recruit MAX and increase theacetylation of Histone H3K27, which enhances the transcription ofEGFL7/miR-126 gene and therefore angiogenesis through MAPK and AKTpathways in human ECs.

FIGS. 17A-B: (FIG. 17A) Strategy for generation of lncEGFL7OS knockoutECs using CRISPR technology. Positions of guide RNAs to delete Exon Iand Exon II of lncEGFL7OS were shown; (FIG. 17B) Efficient deletion ofExon I and/or Exon II in 293T cells using the strategy in FIG. 17A. *indicate the bands after exon I and/or II deletion.

DETAILED DESCRIPTION OF THE DISCLOSURE

Detailed descriptions of one or more preferred embodiments are providedherein. It is to be understood, however, that the present disclosure maybe embodied in various forms. Therefore, specific details disclosedherein are not to be interpreted as limiting, but rather as a basis forthe claims and as a representative basis for teaching one skilled in theart to employ the present disclosure in any appropriate manner.

Wherever any of the phrases “for example,” “such as,” “including” andthe like are used herein, the phrase “and without limitation” isunderstood to follow unless explicitly stated otherwise. Similarly “anexample,” “exemplary” and the like are understood to be non-limiting.

The term “substantially” allows for deviations from the descriptor thatdo not negatively impact the intended purpose. Descriptive terms areunderstood to be modified by the term “substantially” even if the word“substantially” is not explicitly recited. Therefore, for example, thephrase “wherein the lever extends vertically” means “wherein the leverextends substantially vertically” so long as a precise verticalarrangement is not necessary for the lever to perform its function. Theterms “comprising” and “including” and “having” and “involving” (andsimilarly “comprises”, “includes,” “has,” and “involves”) and the likeare used interchangeably and have the same meaning. Specifically, eachof the terms is defined consistent with the common United States patentlaw definition of “comprising” and is therefore interpreted to be anopen term meaning “at least the following,” and is also interpreted notto exclude additional features, limitations, aspects, etc. Thus, forexample, “a process involving steps a, b, and c” means that the processincludes at least steps a, b and c. Wherever the terms “a” or “an” areused, “one or more” is understood, unless such interpretation isnonsensical in context.

I. lncRNAs

A. Background

Long non-coding RNAs (long ncRNAs, lncRNA) are defined as non-proteincoding transcripts longer than 200 nucleotides. This somewhat arbitrarylimit distinguishes long ncRNAs from small regulatory RNAs such asmicroRNAs (miRNAs), short interfering RNAs (siRNAs), Piwi-interactingRNAs (piRNAs), small nucleolar RNAs (snoRNAs), and other short RNAs.However, very recent research has shown that some lncRNAs do encodeproteins.

The current landscape of the mammalian genome is described as numerous‘foci’ of transcription that are separated by long stretches ofintergenic space. While long ncRNAs are located and transcribed withinthe intergenic stretches, the majority are transcribed as complex,interlaced networks of overlapping sense and antisense transcripts thatoften includes protein-coding genes. Genomic sequences within thesetranscriptional foci are often shared within a number of differentcoding and non-coding transcripts in the sense and antisense directionsgiving rise to a complex hierarchy of overlapping isoforms. For example,3012 out of 8961 cDNAs previously annotated as truncated codingsequences within FANTOM2 were later designated as genuine ncRNA variantsof protein-coding cDNAs. While the abundance and conservation of theseinterleaved arrangements suggest they have biological relevance, thecomplexity of these foci frustrates easy evaluation.

The GENCODE consortium has collated and analysed a comprehensive set ofhuman lncRNA annotations and their genomic organisation, modifications,cellular locations and tissue expression profiles. Their analysisindicates human lncRNAs show a bias toward two-exon transcripts.

Many small RNAs, such as microRNAs or snoRNAs, exhibit strongconservation across diverse species. In contrast, long ncRNAs (such asAir and Xist) lack strong conservation, suggesting non-functionality orthe effects of different selection pressures. Unlike mRNAs, which haveto conserve the codon usage and prevent frameshift mutations in a singlelong ORF, selection may conserve only short regions of long ncRNAs thatare constrained by structure or sequence-specific interactions.Therefore, we may see selection act only over small regions of the longncRNA transcript. Thus, despite low conservation of long ncRNAs ingeneral, it should be noted that many long ncRNAs still contain stronglyconserved elements. For example, 19% of highly conserved phastConselements occur in known introns, and another 32% in unannotated regions.Furthermore, a representative set of human long ncRNAs exhibit small,yet significant, reductions in substitution and insertion/deletion ratesindicative of purifying selection that conserve the integrity of thetranscript at the levels of sequence, promoter and splicing.

On the other hand, the low conservation of some ncRNAs may actually bethe result of recent and rapid adaptive selection. For instance, somencRNAs may even be more pliant to evolutionary pressures thanprotein-coding genes, as evidenced by the existence of many lineagespecific ncRNAs, such as the aforementioned Xist or Air. Indeed, thoseconserved regions of the human genome that are subject to recentevolutionary change relative to the chimpanzee genome occurs mainly innon-coding regions, many of which are transcribed. This includes ancRNA, HAR1F, which has undergone rapid evolutionary change in humansand is specifically expressed in the Cajal-Retzius cells in the humanneocortex. The observation that many functionally validated RNAs areevolving quickly may result from these sequences having looserstructure-function constraints, allowing greater evolutionaryinnovation. This is supported by the existence of thousands of sequencesin the mammalian genome that show poor conservation at the primarysequence level but have evidence of conserved RNA secondary structures.

Large-scale sequencing of cDNA libraries and more recentlytranscriptomic sequencing by next generation sequencing indicate thatlong noncoding RNAs number in the order of tens of thousands in mammals.However, despite accumulating evidence suggesting that the majority ofthese are likely to be functional, only a relatively small proportionhas been demonstrated to be biologically relevant. As of January 2016,294 LncRNAs have been functionally annotated in LncRNAdb (a database ofliterature described LncRNAs), with the majority of these (183 LncRNAs)being described in humans. A further large-scale sequencing studyprovides evidence that many transcripts thought to be LncRNAs may, infact, be translated into proteins.

B. lncEGFL7OS

As described below, in an effort to identify potentialangiogenesis-related lncRNAs, the inventors set up a screening forlncRNAs enriched in several EC lines compared to non-ECs. The inventors˜500 EC-enriched lncRNAs, one of which is lncEGFL7OS. This lncRNA islocated in the antisense strand of the EGFL7/miR-126 gene. lncEGFL7OS isa primate-specific lncRNA, and its expression is highly specific to ECsand vascularized tissues. The inventors found that silencing oflncEGFL7OS results in G1 arrest and represses both EC proliferation andmigration. The requirement of lncEGFL7OS for angiogenesis is alsodemonstrated by impaired tube formation in Matrigel assay in vitro andin vivo and repressed vasculogenesis/angiogenesis in an EC/fibroblastco-culture assay upon lncEGFL7OS knockdown.

Moreover, the inventors have developed a human choroid sprouting assaysystem, and found that silencing of lncEGFL7OS significantly represseshuman choroid sprouting ex vivo. Mechanistically, lncEGFL7OS functionsas an enhancer lncRNA that regulates EGFL7/miR-126 expression in ECsthrough repressing DNA methylation in the EGFL7/miR-126 promoter. Takentogether, the inventors have identified primate-specific EC-enrichedlncRNAs, including lncEGFL7OS that are critical for angiogenesis inhumans. The present disclosure provides compositions and methods oftreatment for therapeutics of vascular disorders in humans. Vasculardisorders that can be treated with the present disclosure include tumorgrowth and metastasis associated with cancers, age-related maculardegeneration (AMD), diabetic retinopathy, psoriasis, arthritis, ischemicheart disease, neurodegeneration, hypertension, respiratory distress,and atherosclerosis and other inflammatory diseases.

lncEGFL7OS is deposited as RP11-251M1.1. The sequence is as below:

(SEQ ID NO: 1) TGGGCTCAGGCCCAGAGTGCCAGCTTTGCCCTATCCCATAGCCTGGAGCCACCACAGGAGGGGCACTCCACTCTCTTGGGCTCCTGGAGCCTCAGAGGCAGAGCCAGCCGGGAGTGCAGGAGGGAGAACTTTCCTGTGGACGTCCTGTGTTCTCCAGACGCAGAGAACCCTCATCAACCGAGGGGGAGGTCACTTCCGAATCCACAGATGGCGTGTGAGTGCATGGCGAGCGCCTCCAGGACACACTTACTGTTCCCTTGCTCTGGCCAGACGCCAGCCGGACCCTGTGTGTGCGCGCCGTGCTGCTCTTTGCAGCTGCCTGCAAGGGGTTCCTGCGAAGACCAGCACCTTGGGGAAGAGCCTGCGGCTGAACTTGAACTCGCAGCTACCTGAGTCAGACCTGTGCTTTTTCACCTCTACGGAAGATGTCAGAGCGTTTCCCTAGCAATGTTTTAGAAGTTACTTCTGTCTGGAAAAAAATGGAAAAAATGGCAAATTATGTTATGTATAATTTGATAATTTTAAAGAATTAATGATGTAATTATTACTC AAACCCA

C. Agonists and Antagonists of lncEGFL7OS

Agonists of lncEGFL7OS will generally take one of three forms. First,there is lncEGFL7OS itself. Such molecules may be delivered to targetcells, for example, by injection or infusion, optionally in a deliveryvehicle such as a lipid, such as a liposome or lipid emulsion. Second,one may use expression vectors that drive the expression of lncEGFL7OS.The composition and construction of various expression vectors isdescribed elsewhere in the document. Third, one may use agents distinctfrom lncEGFL7OS that act up-regulate, stabilize or otherwise enhance theactivity of lncEGFL7OS, including small molecules. Such moleculesinclude “mimetics,” molecules which mimic the function, and possiblyform of lncEGFL7OS, but are distinct in chemical structure.

Inhibition of lncRNA function may be achieved by administering antisenseoligonucleotides. The antisense oligonucleotides may be ribonucleotidesor deoxyribonucleotides. Preferably, the antisense oligonucleotides haveat least one chemical modification. Antisense oligonucleotides may becomprised of one or more “locked nucleic acids.” “Locked nucleic acids”(LNAs) are modified ribonucleotides that contain an extra bridge betweenthe 2′ and 4′ carbons of the ribose sugar moiety resulting in a “locked”conformation that confers enhanced thermal stability to oligonucleotidescontaining the LNAs. Alternatively, the antisense oligonucleotides maycomprise peptide nucleic acids (PNAs), which contain a peptide-basedbackbone rather than a sugar-phosphate backbone. Other chemicalmodifications that the antisense oligonucleotides may contain include,but are not limited to, sugar modifications, such as 2′-O-alkyl (e.g.,2′-O-methyl, 2′-O-methoxyethyl), 2′-fluoro, and 4′ thio modifications,and backbone modifications, such as one or more phosphorothioate,morpholino, or phosphonocarboxylate linkages (see, for example, U.S.Pat. Nos. 6,693,187 and 7,067,641, which are herein incorporated byreference in their entireties). In some embodiments, suitable antisenseoligonucleotides are 2′-O-methoxyethyl “gapmers” which contain2′-O-methoxyethyl-modified ribonucleotides on both 5′ and 3′ ends withat least ten deoxyribonucleotides in the center. These “gapmers” arecapable of triggering RNase H-dependent degradation mechanisms of RNAtargets. Other modifications of antisense oligonucleotides to enhancestability and improve efficacy, such as those described in U.S. Pat. No.6,838,283, which is herein incorporated by reference in its entirety,are known in the art and are suitable for use in the methods of thedisclosure. Particular antisense oligonucleotides useful for inhibitingthe activity of lncRNAs are about 19 to about 25 nucleotides in length.Antisense oligonucleotides may comprise a sequence that is at leastpartially complementary to a mature lncRNA sequence, e.g., at leastabout 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to anlncRNA sequence. In some embodiments, the antisense oligonucleotide maybe substantially complementary to an lncRNA sequence, that is at leastabout 95%, 96%, 97%, 98%, or 99% complementary to a targetpolynucleotide sequence. In one embodiment, the antisenseoligonucleotide comprises a sequence that is 100% complementary to anlncRNA sequence.

Another approach for inhibiting the function of lncEGFL7OS isadministering an inhibitory RNA molecule having at least partialsequence identity to the mature lncEGFL7OS sequence. The inhibitory RNAmolecule may be a double-stranded, small interfering RNA (siRNA) or ashort hairpin RNA molecule (shRNA) comprising a stem-loop structure. Thedouble-stranded regions of the inhibitory RNA molecule may comprise asequence that is at least partially identical, e.g., about 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical, to the lncRNA sequence.In some embodiments, the double-stranded regions of the inhibitory RNAcomprise a sequence that is at least substantially identical to thelncRNA sequence. “Substantially identical” refers to a sequence that isat least about 95%, 96%, 97%, 98%, or 99% identical to a targetpolynucleotide sequence. In other embodiments, the double-strandedregions of the inhibitory RNA molecule may contain 100% identity to thetarget lncRNA sequence.

Another approach to regulating lncEGFL7OS is through the design ofCRISPR guide RNAs that target lncEGFL7OS genomic DNA, thereby silencinglncEGFL7OS expression. CRISPRs (clustered regularly interspaced shortpalindromic repeats) are DNA loci containing short repetitions of basesequences. Each repetition is followed by short segments of “spacer DNA”from previous exposures to a virus. CRISPRs are found in approximately40% of sequenced eubacteria genomes and 90% of sequenced archaea.CRISPRs are often associated with cas genes that code for proteinsrelated to CRISPRs. The CRISPR/Cas system is a prokaryotic immune systemthat confers resistance to foreign genetic elements such as plasmids andphages and provides a form of acquired immunity. CRISPR spacersrecognize and silence these exogenous genetic elements like RNAi ineukaryotic organisms.

CRISPR was first shown to work as a genome engineering/editing tool inhuman cell culture by 2012 It has since been used in a wide range oforganisms including baker's yeast (S. cerevisiae), zebra fish, nematodes(C. elegans), plants, mice, and several other organisms. AdditionallyCRISPR has been modified to make programmable transcription factors thatallow scientists to target and activate or silence specific genes.Libraries of tens of thousands of guide RNAs are now available.

CRISPR repeats range in size from 24 to 48 base pairs. They usually showsome dyad symmetry, implying the formation of a secondary structure suchas a hairpin, but are not truly palindromic. Repeats are separated byspacers of similar length. Some CRISPR spacer sequences exactly matchsequences from plasmids and phages, although some spacers match theprokaryote's genome (self-targeting spacers). New spacers can be addedrapidly in response to phage infection.

CRISPR-associated (cas) genes are often associated with CRISPRrepeat-spacer arrays. As of 2013, more than forty different Cas proteinfamilies had been described. Of these protein families, Cas1 appears tobe ubiquitous among different CRISPR/Cas systems. Particularcombinations of cas genes and repeat structures have been used to define8 CRISPR subtypes (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, andMtube), some of which are associated with an additional gene moduleencoding repeat-associated mysterious proteins (RAMPs). More than oneCRISPR subtype may occur in a single genome. The sporadic distributionof the CRISPR/Cas subtypes suggests that the system is subject tohorizontal gene transfer during microbial evolution.

Exogenous DNA is apparently processed by proteins encoded by Cas genesinto small elements (˜30 base pairs in length), which are then somehowinserted into the CRISPR locus near the leader sequence. RNAs from theCRISPR loci are constitutively expressed and are processed by Casproteins to small RNAs composed of individual, exogenously-derivedsequence elements with a flanking repeat sequence. The RNAs guide otherCas proteins to silence exogenous genetic elements at the RNA or DNAlevel. Evidence suggests functional diversity among CRISPR subtypes. TheCse (Cas subtype Ecoli) proteins (called CasA-E in E. coli) form afunctional complex, Cascade, that processes CRISPR RNA transcripts intospacer-repeat units that Cascade retains. In other prokaryotes, Cas6processes the CRISPR transcripts. Interestingly, CRISPR-based phageinactivation in E. coli requires Cascade and Cas3, but not Cas1 andCas2. The Cmr (Cas RAMP module) proteins found in Pyrococcus furiosusand other prokaryotes form a functional complex with small CRISPR RNAsthat recognizes and cleaves complementary target RNAs. RNA-guided CRISPRenzymes are classified as type V restriction enzymes.

See also U.S. Patent Publication 2014/0068797, which is incorporated byreference in its entirety.

Cas9 is a nuclease, an enzyme specialized for cutting DNA, with twoactive cutting sites, one for each strand of the double helix. The teamdemonstrated that they could disable one or both sites while preservingCas9's ability to home located its target DNA. Jinek et al. (2012)combined tracrRNA and spacer RNA into a “single-guide RNA” moleculethat, mixed with Cas9, could find and cut the correct DNA targets. Jinekand coworkers proposed that such synthetic guide RNAs might be able tobe used for gene editing.

Cas9 proteins are highly enriched in pathogenic and commensal bacteria.CRISPR/Cas-mediated gene regulation may contribute to the regulation ofendogenous bacterial genes, particularly during bacterial interactionwith eukaryotic hosts. For example, Cas protein Cas9 of Francisellanovicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) torepress an endogenous transcript encoding a bacterial lipoprotein thatis critical for F. novicida to dampen host response and promotevirulence. Wang et al. showed that coinjection of Cas9 mRNA and sgRNAsinto the germline (zygotes) generated nice with mutations. Delivery ofCas9 DNA sequences also is contemplated.

Clustered Regularly Interspaced Short Palindromic Repeats fromPrevotella and Francisella 1 or CRISPR/Cpf1 is a DNA-editing technologyanalogous to the CRISPR/Cas9 system. Cpf1 is an RNA-guided endonucleaseof a class II CRISPR/Cas system. This acquired immune mechanism is foundin Prevotella and Francisella bacteria. It prevents genetic damage fromviruses. Cpf1 genes are associated with the CRISPR locus, coding for anendonuclease that use a guide RNA to find and cleave viral DNA. Cpf1 isa smaller and simpler endonuclease than Cas9, overcoming some of theCRISPR/Cas9 system limitations. CRISPR/Cpf1 could have multipleapplications, including treatment of genetic illnesses and degenerativeconditions.

As a RNA guided protein, Cas9 (and Cfp1 as well) requires a short RNA todirect the recognition of DNA targets (Mali et al., 2013a). Though Cas9preferentially interrogates DNA sequences containing a PAM sequence NGGit can bind here without a protospacer target. However, the Cas9-gRNAcomplex requires a close match to the gRNA to create a double strandbreak (Cho et al., 2013; Hsu et al., 2013). CRISPR sequences in bacteriaare expressed in multiple RNAs and then processed to create guidestrands for RNA (Bikard et al., 2013). Because Eukaryotic systems lacksome of the proteins required to process CRISPR RNAs the syntheticconstruct gRNA was created to combine the essential pieces of RNA forCas9 targeting into a single RNA expressed with the RNA polymerase typeIII promoter U6 (Mali et al., 2013a, b). Synthetic gRNAs are slightlyover 100 bp at the minimum length and contain a portion which is targetsthe 20 protospacer nucleotides immediately preceding the PAM sequenceNGG; gRNAs do not contain a PAM sequence.

II. Angiogenesis

Angiogenesis is the physiological process through which new bloodvessels form from pre-existing vessels. In precise usage this isdistinct from vasculogenesis, which is the de novo formation ofendothelial cells from mesoderm cell precursors, and fromneovascularization, although discussions are not always precise. Thefirst vessels in the developing embryo form through vasculogenesis,after which angiogenesis is responsible for most, if not all, bloodvessel growth during development and in disease.

Angiogenesis is a normal and vital process in growth and development, aswell as in wound healing and in the formation of granulation tissue.However, it is also a fundamental step in the transition of tumors froma benign state to a malignant one, leading to the use of angiogenesisinhibitors in the treatment of cancer.

Angiogenesis may be a target for combating diseases characterized byeither poor vascularisation or abnormal vasculature. Application ofspecific compounds that may inhibit or induce the creation of new bloodvessels in the body may help combat such diseases. The presence of bloodvessels where there should be none may affect the mechanical propertiesof a tissue, increasing the likelihood of failure. The absence of bloodvessels in a repairing or otherwise metabolically active tissue mayinhibit repair or other essential functions. Several diseases, such asischemic chronic wounds, are the result of failure or insufficient bloodvessel formation and may be treated by a local expansion of bloodvessels, thus bringing new nutrients to the site, facilitating repair.Other diseases, such as age-related macular degeneration, may be createdby a local expansion of blood vessels, interfering with normalphysiological processes.

The modern clinical application of the principle of angiogenesis can bedivided into two main areas: anti-angiogenic therapies, which angiogenicresearch began with, and pro-angiogenic therapies. Whereasanti-angiogenic therapies are being employed to fight cancer andmalignancies, which require an abundance of oxygen and nutrients toproliferate, pro-angiogenic therapies are being explored as options totreat cardiovascular diseases, the number one cause of death in theWestern world. One of the first applications of pro-angiogenic methodsin humans was a German trial using fibroblast growth factor 1 (FGF-1)for the treatment of coronary artery disease. Clinical research intherapeutic angiogenesis is ongoing for a variety of atheroscleroticdiseases, like coronary heart disease, peripheral arterial disease,wound healing disorders, etc.

Also, regarding the mechanism of action, pro-angiogenic methods can bedifferentiated into three main categories: gene-therapy, targeting genesof interest for amplification or inhibition; protein-therapy, whichprimarily manipulates angiogenic growth factors like FGF-1 or vascularendothelial growth factor, VEGF; and cell-based therapies, which involvethe implantation of specific cell types.

There are still serious, unsolved problems related to gene therapy.Difficulties include effective integration of the therapeutic genes intothe genome of target cells, reducing the risk of an undesired immuneresponse, potential toxicity, immunogenicity, inflammatory responses,and oncogenesis related to the viral vectors used in implanting genesand the sheer complexity of the genetic basis of angiogenesis. The mostcommonly occurring disorders in humans, such as heart disease, highblood pressure, diabetes and Alzheimer's disease, are most likely causedby the combined effects of variations in many genes, and, thus,injecting a single gene may not be significantly beneficial in suchdiseases.

In contrast, pro-angiogenic protein therapy uses well-defined, preciselystructured proteins, with previously defined optimal doses of theindividual protein for disease states, and with well-known biologicaleffects. On the other hand, an obstacle of protein therapy is the modeof delivery. Oral, intravenous, intra-arterial, or intramuscular routesof protein administration are not always as effective, as thetherapeutic protein may be metabolized or cleared before it can enterthe target tissue. Cell-based pro-angiogenic therapies are still earlystages of research, with many open questions regarding best cell typesand dosages to use.

III. Methods of Treating Disease States

The present disclosure provides methods of treating various diseasestates by administering to a subject agonists or antagonists oflncEGFL7OS. For the purposes of the present application, treatmentcomprises reducing one or more of the symptoms of associated with thedisease states discussed below. Any level of improvement will beconsidered treatment, and there is no requirement for a particular levelof improvement or a “cure.” It is also sufficient in treatment thatsymptoms be stabilized, i.e., that the disease condition will notworsen.

The present disclosure provides a method of promoting vascular integrityand/or vascular repair comprising administering to a subject sufferingfrom a vascular condition an agonist of lncEGFL7OS function. Thevascular condition may include, but is not limited to, myocardialinfarction, ischemia-reperfusion injury, stenosis, fibrosis, vasculartrauma, vascular leakage, psoriasis, arthritis, neurodegeneration,hypertension, and respiratory distress.

A. Conditions Impairing Vascular Integrity or Causing the Need forVascular Repair

As discussed above, the present disclosure provides for the use ofagonists of lncEGFL7OS to improve the integrity of vascular tissue, andalso to promote vascular repair and neovascularization following injury,including ischemic insults. The following disease states/conditions arespecifically contemplated for treatment according to the presentdisclosure, but are not limiting.

Treatment regimens would vary depending on the clinical situation.However, long-term maintenance would appear to be appropriate in mostcircumstances. It also may be desirable treat vascular conditions withmodulators of lncEGFL7OS intermittently, such as within a brief windowduring disease progression.

Myocardial Infarction.

Myocardial infarction (MI), occurs when the blood supply to part of theheart is interrupted. This is most commonly due to occlusion (blockage)of a coronary artery following the rupture of a vulnerableatherosclerotic plaque, which is an unstable collection of lipids (likecholesterol) and white blood cells (especially macrophages) in the wallof an artery. The resulting ischemia (restriction in blood supply) andoxygen shortage, if left untreated for a sufficient period, can causedamage and/or death (infarction) of heart muscle tissue (myocardium).

Classical symptoms of acute myocardial infarction (AMI) include suddenchest pain (typically radiating to the left arm or left side of theneck), shortness of breath, nausea, vomiting, palpitations, sweating,and anxiety (often described as a sense of impending doom). Women mayexperience fewer typical symptoms than men, most commonly shortness ofbreath, weakness, a feeling of indigestion, and fatigue. Approximatelyone quarter of all myocardial infarctions are silent, without chest painor other symptoms. A heart attack is a medical emergency, and peopleexperiencing chest pain are advised to alert their emergency medicalservices, because prompt treatment is beneficial.

Immediate treatment for suspected acute myocardial infarction includesoxygen, aspirin, and sublingual glyceryl trinitrate (colloquiallyreferred to as nitroglycerin and abbreviated as NTG or GTN). Pain reliefis also often given, classically morphine sulfate. The patient willreceive a number of diagnostic tests, such as an electrocardiogram (ECG,EKG), a chest X-ray and blood tests to detect elevations in cardiacmarkers (blood tests to detect heart muscle damage). The most often usedmarkers are the creatine kinase-MB (CK-MB) fraction and the troponin I(TnI) or troponin T (TnT) levels. On the basis of the ECG, a distinctionis made between ST elevation MI (STEMI) or non-ST elevation MI (NSTEMI).Most cases of STEMI are treated with thrombolysis or if possible withpercutaneous coronary intervention (PCI, angioplasty and stentinsertion), provided the hospital has facilities for coronaryangiography. NSTEMI is managed with medication, although PCI is oftenperformed during hospital admission. In patients who have multipleblockages and who are relatively stable, or in a few extraordinaryemergency cases, bypass surgery of the blocked coronary artery is anoption.

Ischemia-Reperfusion Injury.

Ischemia-reperfusion injury is caused at least in part by theinflammatory response of damaged tissues. White blood cells carried tothe area by the newly returning blood release a host of inflammatoryfactors such as interleukins as well as free radicals in response totissue damage. The restored blood flow reintroduces oxygen within cellsthat damages cellular proteins, DNA and the plasma membrane. Damage tothe cell's membrane may in turn cause the release of more free radicals.Such reactive species may also act indirectly in redox signaling to turnon apoptosis. Leukocytes may also build up in small capillaries,obstructing them and leading to more ischemia.

Reperfusion injury plays a part in the brain's ischemic cascade, whichis involved in stroke and brain trauma. Repeated bouts of ischemia andreperfusion injury also are thought to be a factor leading to theformation and failure to heal of chronic wounds such as pressure soresand diabetic foot ulcers. Continuous pressure limits blood supply andcauses ischemia, and the inflammation occurs during reperfusion. As thisprocess is repeated, it eventually damages tissue enough to cause awound.

Glisodin, a dietary supplement derived from superoxide dismutase (SOD)and wheat gliadin, has been studied for its ability to mitigateischemia-reperfusion injury. A study of aortic cross-clamping, a commonprocedure in cardiac surgery, demonstrated a strong potential benefitwith further research ongoing.

Stenosis.

A stenosis is an abnormal narrowing in a blood vessel or other tubularorgan or structure. Stenoses of the vascular type are often associatedwith a noise (bruit) resulting from turbulent flow over the narrowedblood vessel. This bruit can be made audible by a stethoscope. Other,more reliable methods of diagnosing a stenosis are imaging methodsincluding ultrasound, Magnetic Resonance Imaging/Magnetic ResonanceAngiography, Computed Tomography/CT-Angiography which combine anatomicimaging (i.e., the visible narrowing of a vessel) with the display offlow phenomena (visualization of the movement of the bodily fluidthrough the bodily structure). Vascular stenoses include intermittentclaudication (peripheral artery stenosis), angina (coronary arterystenosis), carotid artery stenosis which predispose to (strokes andtransient ischemic episodes) and renal artery stenosis.

Other Conditions.

Trauma and vascular leakage are also conditions which may be treatedwith lncEGFL7OS or agonists thereof.

Risks.

The present disclosure also contemplates treating individuals at riskfor any of the aforementioned disease states. These individuals wouldinclude those persons suffering from fibrosis. hypertension, cardiachypertrophy, osteoporosis, neurodegeneration, and/or respiratorydistress.

B. Pathologic Neovascularization

As discussed above, the present disclosure provides for the use ofantagonists of lncEGFL7OS to impede neovasculariziation that leads to orcontributes to disease. The following disease states/conditions arespecifically contemplated for treatment according to the presentdisclosure, but are not limiting.

The present disclosure provides a method of inhibiting pathologicvascularization in a subject in need thereof comprising administering toa subject an antagonist of lncEGFL7OS. A condition associated withpathologic vascularization includes, but is not limited to,atherosclerosis, retinopathy, cancer, and stroke.

Early Stage Atherosclerosis.

Atherosclerosis is a disease affecting arterial blood vessels. It is achronic inflammatory response in the walls of arteries, in large partdue to the accumulation of macrophage white blood cells and promoted bylow density (especially small particle) lipoproteins (plasma proteinsthat carry cholesterol and triglycerides) without adequate removal offats and cholesterol from the macrophages by functional high densitylipoproteins (HDL). It is commonly referred to as a “hardening” of thearteries. It is caused by the formation of multiple plaques within thearteries.

Atherosclerosis develops from low-density lipoprotein cholesterol (LDL),colloquially called “bad cholesterol.” When this lipoprotein getsthrough the wall of an artery, oxygen free radicals react with it toform oxidized-LDL. The body's immune system responds by sendingspecialised white blood cells (macrophages and T-lymphocytes) to absorbthe oxidized-LDL. Unfortunately, these white blood cells are not able toprocess the oxidized-LDL, and ultimately grow then rupture, depositing agreater amount of oxidized cholesterol into the artery wall. Thistriggers more white blood cells, continuing the cycle. Eventually, theartery becomes inflamed. The cholesterol plaque causes the muscle cellsto enlarge and form a hard cover over the affected area. This hard coveris what causes a narrowing of the artery, reduces the blood flow andincreases blood pressure.

Atherosclerosis typically begins in early adolescence, and is usuallyfound in most major arteries, yet is asymptomatic and not detected bymost diagnostic methods during life. The stage immediately prior toactual atherosclerosis is known as subclinical atherosclerosis. It mostcommonly becomes seriously symptomatic when interfering with thecoronary circulation supplying the heart or cerebral circulationsupplying the brain, and is considered the most important underlyingcause of strokes, heart attacks, various heart diseases includingcongestive heart failure, and most cardiovascular diseases, in general.Atheroma in arm, or more often in leg arteries, which produces decreasedblood flow is called Peripheral artery occlusive disease (PAOD). Mostartery flow disrupting events occur at locations with less than 50%lumen narrowing (˜20% stenosis is average).

Although the disease process tends to be slowly progressive overdecades, it usually remains asymptomatic until an atheroma obstructs thebloodstream in the artery. This is typically by rupture of an atheroma,clotting and fibrous organization of the clot within the lumen, coveringthe rupture but also producing stenosis, or over time and after repeatedruptures, resulting in a persistent, usually localized stenosis.Stenoses can be slowly progressive, whereas plaque rupture is a suddenevent that occurs specifically in atheromas with thinner/weaker fibrouscaps that have become “unstable.”

Repeated plaque ruptures, ones not resulting in total lumen closure,combined with the clot patch over the rupture and healing response tostabilize the clot, is the process that produces most stenoses overtime. The stenotic areas tend to become more stable, despite increasedflow velocities at these narrowings. Most major blood-flow-stoppingevents occur at large plaques, which, prior to their rupture, producedvery little if any stenosis.

From clinical trials, 20% is the average stenosis at plaques thatsubsequently rupture with resulting complete artery closure. Most severeclinical events do not occur at plaques that produce high-gradestenosis. From clinical trials, only 14% of heart attacks occur fromartery closure at plaques producing a 75% or greater stenosis prior tothe vessel closing.

If the fibrous cap separating a soft atheroma from the bloodstreamwithin the artery ruptures, tissue fragments are exposed and released,and blood enters the atheroma within the wall and sometimes results in asudden expansion of the atheroma size. Tissue fragments are veryclot-promoting, containing collagen and tissue factor; they activateplatelets and activate the system of coagulation. The result is theformation of a thrombus (blood clot) overlying the atheroma, whichobstructs blood flow acutely. With the obstruction of blood flow,downstream tissues are starved of oxygen and nutrients. If this is themyocardium (heart muscle), angina (cardiac chest pain) or myocardialinfarction (heart attack) develops.

If atherosclerosis leads to symptoms, some symptoms such as anginapectoris can be treated. Non-pharmaceutical means are usually the firstmethod of treatment, such as cessation of smoking and practicing regularexercise. If these methods do not work, medicines are usually the nextstep in treating cardiovascular diseases, and, with improvements, haveincreasingly become the most effective method over the long term.However, medicines are criticized for their expense, patented controland occasional undesired effects.

In general, the group of medications referred to as statins has been themost popular and are widely prescribed for treating atherosclerosis.They have relatively few short-term or longer-term undesirableside-effects, and multiple comparative treatment/placebo trials havefairly consistently shown strong effects in reducing atheroscleroticdisease ‘events’ and generally ˜25% comparative mortality reduction inclinical trials, although one study design, ALLHAT, was less stronglyfavorable.

The newest statin, rosuvastatin, has been the first to demonstrateregression of atherosclerotic plaque within the coronary arteries byIVUS (intravascular ultrasound evaluation), The study was set up todemonstrate effect primarily on atherosclerosis volume within a 2 yeartime-frame in people with active/symptomatic disease (angina frequencyalso declined markedly) but not global clinical outcomes, which wasexpected to require longer trial time periods; these longer trialsremain in progress.

However, for most people, changing their physiologic behaviors, from theusual high risk to greatly reduced risk, requires a combination ofseveral compounds, taken on a daily basis and indefinitely. More andmore human treatment trials have been done and are ongoing thatdemonstrate improved outcome for those people using more-complex andeffective treatment regimens that change physiologic behaviour patternsto more closely resemble those that humans exhibit in childhood at atime before fatty streaks begin forming.

Retinopathy.

Retinopathy is a general term that refers to some form ofnon-inflammatory damage to the retina of the eye. Most commonly it is aproblem with the blood supply that is the cause for this condition.Frequently, retinopathy is an ocular manifestation of systemic disease.Retinopathy is diagnosed by an optometrist or an ophthalmologist duringophthalmoscopy. Treatment depends on the cause of the disease.

The main causes of retinopathy are diabetes—diabetic retinopathy;arterial hypertension—hypertensive retinopathy; prematurity of thenewborn—retinopathy of prematurity (ROP); sickle cell anemia; geneticretinopathy; direct sunlight exposure—solar retinopathy; medicinalproducts—drug-related retinopathy; and retinal vein or artery occlusion.Many types of retinopathy are progressive and may result in blindness orsevere vision loss or impairment, particularly if the macula becomesaffected.

Age-Related Macular Degeneration.

Macular degeneration, also known as age-related macular degeneration(AMD or ARMD), is a medical condition which may result in blurred or novision in the center of the visual field. Early on there are often nosymptoms. Over time, however, some people experience a gradual worseningof vision that may affect one or both eyes. While it does not result incomplete blindness, loss of central vision can make it hard to recognizefaces, drive, read, or perform other activities of daily life. Visualhallucinations may also occur but these do not represent a mentalillness.

Macular degeneration typically occurs in older people. Genetic factorsand smoking also play a role. It is due to damage to the macula of theretina. Diagnosis is by a complete eye exam. The severity is dividedinto early, intermediate, and late types. The late type is additionallydivided into “dry” and “wet” forms with the dry form making up 90% ofcases.

Prevention includes exercising, eating well, and not smoking.Antioxidant vitamins and minerals do not appear to be useful forprevention. There is no cure or treatment that returns vision alreadylost. In the wet form, anti-VEGF medication injected into the eye orless commonly laser coagulation or photodynamic therapy may slowworsening. Supplements in those who already have the disease may slowprogression.

In 2010 it affected 23.5 million people globally. In 2013 moderate tosevere disease affected 13.4 million and it is the fourth most commoncause of blindness after cataracts, preterm birth, and glaucoma. It mostcommonly occurs in people over the age of fifty and in the United Statesis the most common cause of vision loss in this age group. About 0.4% ofpeople between 50 and 60 have the disease, while it occurs in 0.7% ofpeople 60 to 70, 2.3% of those 70 to 80, and nearly 12% of people over80 years old.

Signs and symptoms of macular degeneration include (a) distorted visionin the form of metamorphopsia, in which a grid of straight lines appearswavy and parts of the grid may appear blank: Patients often first noticethis when looking at things like miniblinds in their home or telephonepoles while driving. There may also be central scotomas, shadows ormissing areas of vision; (b) slow recovery of visual function afterexposure to bright light (photostress test); (c) visual acuitydrastically decreasing (two levels or more), e.g., 20/20 to 20/80; (d)blurred vision: Those with nonexudative macular degeneration may beasymptomatic or notice a gradual loss of central vision, whereas thosewith exudative macular degeneration often notice a rapid onset of visionloss (often caused by leakage and bleeding of abnormal blood vessels);(e) trouble discerning colors, specifically dark ones from dark ones andlight ones from light ones; (f) a loss in contrast sensitivity.

Macular degeneration by itself will not lead to total blindness. Forthat matter, only a very small number of people with visual impairmentare totally blind. In almost all cases, some vision remains, mainlyperipheral. Other complicating conditions may possibly lead to such anacute condition (severe stroke or trauma, untreated glaucoma, etc.), butfew macular degeneration patients experience total visual loss.

The area of the macula comprises only about 2.1% of the retina, and theremaining 97.9% (the peripheral field) remains unaffected by thedisease. Even though the macula provides such a small fraction of thevisual field, almost half of the visual cortex is devoted to processingmacular information.

The loss of central vision profoundly affects visual functioning. It isquite difficult, for example, to read without central vision. Picturesthat attempt to depict the central visual loss of macular degenerationwith a black spot do not really do justice to the devastating nature ofthe visual loss. This can be demonstrated by printing letters six incheshigh on a piece of paper and attempting to identify them while lookingstraight ahead and holding the paper slightly to the side. Most peoplefind this difficult to do.

The pathogenesis of age-related macular degeneration is not well known,although a number of theories have been put forward, including oxidativestress, mitochondrial dysfunction, and inflammatory processes.

The imbalance between production of damaged cellular components anddegradation leads to the accumulation of detrimental products, forexample, intracellular lipofuscin and extracellular drusen. Incipientatrophy is demarcated by areas of retinal pigment epithelium (RPE)thinning or depigmentation that precede geographic atrophy in the earlystages of AMD. In advanced stages of AMD, atrophy of the RPE (geographicatrophy) and/or development of new blood vessels (neovascularization)result in death of photoreceptors and central vision loss.

In the dry (nonexudative) form, cellular debris called drusenaccumulates between the retina and the choroid, causing atrophy andscarring to the retina. In the wet (exudative) form, which is moresevere, blood vessels grow up from the choroid (neovascularization)behind the retina which can leak exudate and fluid and also causehemorrhaging.

Early work demonstrated a family of immune mediators was plentiful indrusen.¹ Complement factor H (CFH) is an important inhibitor of thisinflammatory cascade, and a disease-associated polymorphism in the CFHgene strongly associates with AMD. Thus an AMD pathophysiological modelof chronic low grade complement activation and inflammation in themacula has been advanced. Lending credibility to this has been thediscovery of disease-associated genetic polymorphisms in other elementsof the complement cascade including complement component 3 (C3).

A powerful predictor of AMD is found on chromosome 10q26 at LOC 387715.An insertion/deletion polymorphism at this site reduces expression ofthe ARMS2 gene though destabilization of its mRNA through deletion ofthe polyadenylation signal. ARMS2 protein may localize to themitochondria and participate in energy metabolism, though much remainsto be discovered about its function. Other gene markers of progressionrisk includes tissue inhibitor of metalloproteinase 3 (TIMP3),suggesting a role for intracellular matrix metabolism in AMDprogression. Variations in cholesterol metabolising genes such as thehepatic lipase, cholesterol ester transferase, lipoprotein lipase andthe ABC-binding cassette A1 correlate with disease progression. Theearly stigmata of disease, drusen, are rich in cholesterol, offeringface validity to the results of genome-wide association studies.

Diagnosis of age-related macular degeneration rests on signs in themacula, irrespective of visual acuity. Diagnosis of AMD may include thefollowing procedures and tests. The transition from dry to wet AMD canhappen rapidly, and if it is left untreated can lead to legal blindnessin as little as six months. To prevent this from occurring and toinitiate preventative strategies earlier in the disease process, darkadaptation testing may be performed. A dark adaptometer can detectsubclinical AMD at least three years earlier than it is clinicallyevident.

There is a loss of contrast sensitivity, so that contours, shadows, andcolor vision are less vivid. The loss in contrast sensitivity can bequickly and easily measured by a contrast sensitivity test like PelliRobson performed either at home or by an eye specialist. When viewing anAmsler grid, some straight lines appear wavy and some patches appearblank. When viewing a Snellen chart, at least 2 lines decline.

In dry macular degeneration, which occurs in 85-90 percent of AMD cases,drusen spots can be seen in Fundus photography. In wet maculardegeneration, angiography can visualize the leakage of bloodstreambehind the macula. Fluorescein angiography allows for the identificationand localization of abnormal vascular processes. Using anelectroretinogram, points in the macula with a weak or absent responsecompared to a normal eye may be found Farnsworth-Munsell 100 hue testand Maximum Color Contrast Sensitivity test (MCCS) for assessing coloracuity and color contrast sensitivity. Optical coherence tomography isnow used by most ophthalmologists in the diagnosis and the follow-upevaluation of the response to treatment with antiangiogenic drugs.

In addition to the pigmented cells in the iris (the colored part of theeye), there are pigmented cells beneath the retina. As these cells breakdown and release their pigment, dark clumps of released pigment andlater, areas that are less pigmented may appear. Exudative changes(hemorrhages in the eye, hard exudates, subretinal/sub-RPE/intraretinalfluid) and drusen, tiny accumulations of extracellular material thatbuild up on the retina, also occur. While there is a tendency for drusento be blamed for the progressive loss of vision, drusen deposits can bepresent in the retina without vision loss. Some patients with largedeposits of drusen have normal visual acuity. If normal retinalreception and image transmission are sometimes possible in a retina whenhigh concentrations of drusen are present, then, even if drusen can beimplicated in the loss of visual function, there must be at least oneother factor that accounts for the loss of vision.

Stroke.

Stroke is the rapidly developing loss of brain functions due to adisturbance in the blood vessels supplying blood to the brain. This canbe due to ischemia (lack of blood supply) caused by thrombosis orembolism, or due to a hemorrhage. It can cause permanent neurologicaldamage, complications and death if not promptly diagnosed and treated.Risk factors for stroke include advanced age, hypertension (high bloodpressure), previous stroke or transient ischemic attack (TIA), diabetes,high cholesterol, cigarette smoking, atrial fibrillation,estrogen-containing forms of hormonal contraception, migraine with aura,and thrombophilia (a tendency to thrombosis), patent foramen ovale andseveral rarer disorders. High blood pressure is the most importantmodifiable risk factor of stroke.

The traditional definition of stroke, devised by the World HealthOrganization in the 1970s, is a “neurological deficit of cerebrovascularcause that persists beyond 24 hours or is interrupted by death within 24hours.” The 24-hour limit divides stroke from transient ischemic attack,which is a related syndrome of stroke symptoms that resolve completelywithin 24 hours. With the availability of treatments that, when givenearly, can reduce stroke severity, many now prefer alternative concepts,such as brain attack and acute ischemic cerebrovascular syndrome(modeled after heart attack and acute coronary syndrome respectively),that reflect the urgency of stroke symptoms and the need to act swiftly.

Stroke is occasionally treated with thrombolysis (“clot-buster”), butusually with supportive care (physiotherapy and occupational therapy)and secondary prevention with antiplatelet drugs (aspirin and oftendipyridamole), blood pressure control, statins and anticoagulation (inselected patients).

Strokes can be classified into two major categories: ischemic andhemorrhagic. Ischemia is due to interruption of the blood supply, whilehemorrhage is due to rupture of a blood vessel or an abnormal vascularstructure. 80% of strokes are due to ischemia; the remainder are due tohemorrhage.

In an ischemic stroke, blood supply to part of the brain is decreased,leading to dysfunction and necrosis of the brain tissue in that area.There are four reasons why this might happen: thrombosis (obstruction ofa blood vessel by a blood clot forming locally), embolism (idem due toan embolus from elsewhere in the body, see below), systemichypoperfusion (general decrease in blood supply, e.g., in shock) andvenous thrombosis. Stroke without an obvious explanation is termed“cryptogenic” (of unknown origin).

In thrombotic stroke, a thrombus (blood clot) usually forms aroundatherosclerotic plaques. Since blockage of the artery is gradual, onsetof symptomatic thrombotic strokes is slower. A thrombus itself (even ifnon-occluding) can lead to an embolic stroke (see below) if the thrombusbreaks off, at which point it is called an “embolus.” Thrombotic strokecan be divided into two types depending on the type of vessel thethrombus is formed on—large vessel disease or small vessel disease.

Embolic stroke refers to the blockage of an artery by an embolus, atraveling particle or debris in the arterial bloodstream originatingfrom elsewhere. An embolus is most frequently a thrombus, but it canalso be a number of other substances including fat (e.g. from bonemarrow in a broken bone), air, cancer cells or clumps of bacteria(usually from infectious endocarditis). Because an embolus arises fromelsewhere, local therapy only solves the problem temporarily. Thus, thesource of the embolus must be identified. Because the embolic blockageis sudden in onset, symptoms usually are maximal at start. Also,symptoms may be transient as the embolus is partially resorbed and movesto a different location or dissipates altogether. Emboli most commonlyarise from the heart (especially in atrial fibrillation) but mayoriginate from elsewhere in the arterial tree. In paradoxical embolism,a deep vein thrombosis embolises through an atrial or ventricular septaldefect in the heart into the brain.

Cardiac causes can be distinguished between high- and low-risk:

-   -   High risk: atrial fibrillation and paroxysmal atrial        fibrillation, rheumatic disease of the mitral or aortic valve        disease, artificial heart valves, known cardiac thrombus of the        atrium or ventricle, sick sinus syndrome, sustained atrial        flutter, recent myocardial infarction, chronic myocardial        infarction together with ejection fraction <28 percent,        symptomatic congestive heart failure with ejection fraction <30        percent, dilated cardiomyopathy, Libman-Sacks endocarditis,        Marantic endocarditis, infective endocarditis, papillary        fibroelastoma, left atrial myxoma and coronary artery bypass        graft (CABG) surgery.    -   Low risk/potential: calcification of the annulus (ring) of the        mitral valve, patent foramen ovale (PFO), atrial septal        aneurysm, atrial septal aneurysm with patent foramen ovale, left        ventricular aneurysm without thrombus, isolated left atrial        “smoke” on echocardiography (no mitral stenosis or atrial        fibrillation), complex atheroma in the ascending aorta or        proximal arch.

Systemic hypoperfusion is the reduction of blood flow to all parts ofthe body. It is most commonly due to cardiac pump failure from cardiacarrest or arrhythmias, or from reduced cardiac output as a result ofmyocardial infarction, pulmonary embolism, pericardial effusion, orbleeding. Hypoxemia (low blood oxygen content) may precipitate thehypoperfusion. Because the reduction in blood flow is global, all partsof the brain may be affected, especially “watershed” areas—border zoneregions supplied by the major cerebral arteries. Blood flow to theseareas does not necessarily stop, but instead it may lessen to the pointwhere brain damage can occur. This phenomenon is also referred to as“last meadow” to point to the fact that in irrigation the last meadowreceives the least amount of water.

Cerebral venous sinus thrombosis leads to stroke due to locallyincreased venous pressure, which exceeds the pressure generated by thearteries. Infarcts are more likely to undergo hemorrhagic transformation(leaking of blood into the damaged area) than other types of ischemicstroke.

An ischemic stroke is due to a thrombus (blood clot) occluding acerebral artery, a patient is given antiplatelet medication (aspirin,clopidogrel, dipyridamole), or anticoagulant medication (warfarin),dependent on the cause, when this type of stroke has been found.Hemorrhagic stroke must be ruled out with medical imaging, since thistherapy would be harmful to patients with that type of stroke.

Other immediate strategies to protect the brain during stroke includeensuring that blood sugar is as normal as possible (such as commencementof an insulin sliding scale in known diabetics), and that the strokepatient is receiving adequate oxygen and intravenous fluids. The patientmay be positioned so that his or her head is flat on the stretcher,rather than sitting up, since studies have shown that this increasesblood flow to the brain. Additional therapies for ischemic strokeinclude aspirin (50 to 325 mg daily), clopidogrel (75 mg daily), andcombined aspirin and dipyridamole extended release (25/200 mg twicedaily).

It is common for the blood pressure to be elevated immediately followinga stroke. Studies indicated that while high blood pressure causesstroke, it is actually beneficial in the emergency period to allowbetter blood flow to the brain.

If studies show carotid stenosis, and the patient has residual functionin the affected side, carotid endarterectomy (surgical removal of thestenosis) may decrease the risk of recurrence if performed rapidly afterstroke.

If the stroke has been the result of cardiac arrhythmia with cardiogenicemboli, treatment of the arrhythmia and anticoagulation with warfarin orhigh-dose aspirin may decrease the risk of recurrence. Stroke preventiontreatment for a common arrhythmia, atrial fibrillation, is determinedaccording to the CHADS/CHADS2 system.

In increasing numbers of primary stroke centers, pharmacologicthrombolysis (“clot busting”) with the drug tissue plasminogen activator(tPA), is used to dissolve the clot and unblock the artery. However, theuse of tPA in acute stroke is controversial. Another intervention foracute ischemic stroke is removal of the offending thrombus directly.This is accomplished by inserting a catheter into the femoral artery,directing it into the cerebral circulation, and deploying acorkscrew-like device to ensnare the clot, which is then withdrawn fromthe body. Anticoagulation can prevent recurrent stroke. Among patientswith nonvalvular atrial fibrillation, anticoagulation can reduce strokeby 60% while antiplatelet agents can reduce stroke by 20%. However, arecent meta-analysis suggests harm from anti-coagulation started earlyafter an embolic stroke.

Cancer.

Cancers comprise a class of diseases in which a group of cells displayuncontrolled growth (division beyond the normal limits), invasion(intrusion on and destruction of adjacent tissues), and sometimesmetastasis (spread to other locations in the body via lymph or blood).These three malignant properties of cancers differentiate them frombenign tumors, which are self-limited, do not invade or metastasize.Most cancers form a tumor but some, like leukemia, do not. The branch ofmedicine concerned with the study, diagnosis, treatment, and preventionof cancer is oncology.

Nearly all cancers are caused by abnormalities in the genetic materialof the transformed cells. These abnormalities may be due to the effectsof carcinogens, such as tobacco smoke, radiation, chemicals, orinfectious agents. Other cancer-promoting genetic abnormalities may berandomly acquired through errors in DNA replication, or are inherited,and thus present in all cells from birth. The heritability of cancersare usually affected by complex interactions between carcinogens and thehost's genome. New aspects of the genetics of cancer pathogenesis, suchas DNA methylation, and microRNAs are increasingly recognized asimportant.

Diagnosis usually requires the histologic examination of a tissue biopsyspecimen by a pathologist, although the initial indication of malignancycan be symptoms or radiographic imaging abnormalities. Most cancers canbe treated and some cured, depending on the specific type, location, andstage. Once diagnosed, cancer is usually treated with a combination ofsurgery, chemotherapy and radiotherapy.

Radiation therapy (also called radiotherapy, X-ray therapy, orirradiation) is the use of ionizing radiation to kill cancer cells andshrink tumors. Radiation therapy can be administered externally viaexternal beam radiotherapy (EBRT) or internally via brachytherapy.Radiation therapy may be used to treat almost every type of solid tumor,including cancers of the brain, breast, cervix, larynx, lung, pancreas,prostate, skin, stomach, uterus, or soft tissue sarcomas. Radiation isalso used to treat leukemia and lymphoma. Radiation dose to each sitedepends on a number of factors, including the radiosensitivity of eachcancer type and whether there are tissues and organs nearby that may bedamaged by radiation. Thus, as with every form of treatment, radiationtherapy is not without its side effects.

Chemotherapy is the treatment of cancer with drugs that can destroycancer cells. In current usage, the term “chemotherapy” usually refersto cytotoxic drugs which affect rapidly dividing cells in general, incontrast with targeted therapy (see below). Chemotherapy drugs interferewith cell division in various possible ways, e.g., with the duplicationof DNA or the separation of newly formed chromosomes. Most forms ofchemotherapy target all rapidly dividing cells and are not specific forcancer cells, although some degree of specificity may come from theinability of many cancer cells to repair DNA damage, while normal cellsgenerally can. Hence, chemotherapy has the potential to harm healthytissue, especially those tissues that have a high replacement rate(e.g., intestinal lining). These cells usually repair themselves afterchemotherapy. Because some drugs work better together than alone, two ormore drugs are often given at the same time. This is called “combinationchemotherapy,” and indeed, most chemotherapy regimens are given in acombination.

Targeted therapy, which first became available in the late 1990's, hashad a significant impact in the treatment of some types of cancer, andis currently a very active research area. This constitutes the use ofagents specific for the deregulated proteins of cancer cells. Smallmolecule targeted therapy drugs are generally inhibitors of enzymaticdomains on mutated, overexpressed, or otherwise critical proteins withinthe cancer cell. Prominent examples are the tyrosine kinase inhibitorsimatinib and gefitinib.

Monoclonal antibody therapy is another strategy in which the therapeuticagent is an antibody which specifically binds to a protein on thesurface of the cancer cells. Examples include the anti-HER2/neu antibodytrastuzumab (Herceptin) used in breast cancer, and the anti-CD20antibody rituximab, used in a variety of B-cell malignancies.

Targeted therapy can also involve small peptides as “homing devices”which can bind to cell surface receptors or affected extracellularmatrix surrounding the tumor. Radionuclides which are attached to thispeptides (e.g., RGDs) eventually kill the cancer cell if the nuclidedecays in the vicinity of the cell. Especially oligo- or multimers ofthese binding motifs are of great interest, since this can lead toenhanced tumor specificity and avidity.

Photodynamic therapy (PDT) is a ternary treatment for cancer involving aphotosensitizer, tissue oxygen, and light (often using lasers). PDT canbe used as treatment for basal cell carcinoma (BCC) or lung cancer; PDTcan also be useful in removing traces of malignant tissue after surgicalremoval of large tumors.

Cancer immunotherapy refers to a diverse set of therapeutic strategiesdesigned to induce the patient's own immune system to fight the tumor.Contemporary methods for generating an immune response against tumorsinclude intravesical BCG immunotherapy for superficial bladder cancer,and use of interferons and other cytokines to induce an immune responsein renal cell carcinoma and melanoma patients. Vaccines to generatespecific immune responses are the subject of intensive research for anumber of tumors, notably malignant melanoma and renal cell carcinoma.Sipuleucel-T is a vaccine-like strategy in late clinical trials forprostate cancer in which dendritic cells from the patient are loadedwith prostatic acid phosphatase peptides to induce a specific immuneresponse against prostate-derived cells.

Allogeneic hematopoietic stem cell transplantation (“bone marrowtransplantation” from a genetically non-identical donor) can beconsidered a form of immunotherapy, since the donor's immune cells willoften attack the tumor in a phenomenon known as graft-versus-tumoreffect. For this reason, allogeneic HSCT leads to a higher cure ratethan autologous transplantation for several cancer types, although theside effects are also more severe.

The growth of some cancers can be inhibited by providing or blockingcertain hormones. Common examples of hormone-sensitive tumors includecertain types of breast and prostate cancers. Removing or blockingestrogen or testosterone is often an important additional treatment. Incertain cancers, administration of hormone agonists, such asprogestogens may be therapeutically beneficial.

Angiogenesis inhibitors prevent the extensive growth of blood vessels(angiogenesis) that tumors require to survive. Some, such asbevacizumab, have been approved and are in clinical use. One of the mainproblems with anti-angiogenesis drugs is that many factors stimulateblood vessel growth, in normal cells and cancer. Anti-angiogenesis drugsonly target one factor, so the other factors continue to stimulate bloodvessel growth. Other problems include route of administration,maintenance of stability and activity and targeting at the tumorvasculature.

Risk.

The present disclosure also contemplates treating individuals at riskfor any of the aforementioned disease states. These individuals wouldinclude those persons suffering from atherosclerosis, obesity, asthma,arthritis, psoriasis and/or blindness.

C. Combined Therapy

In another embodiment, it is envisioned to use a modulator of lncEGFL7OSin combination with other therapeutic modalities. Thus, in addition tothe therapies described above, one may also provide to the patient more“standard” pharmaceutical therapies. Combinations may be achieved bycontacting cells, tissues or subjects with a single composition orpharmacological formulation that includes both agents, or by contactingthe cell with two distinct compositions or formulations, at the sametime, wherein one composition includes the expression construct and theother includes the agent. Alternatively, the therapy using a modulatorof lncEGFL7OS may precede or follow administration of the other agent(s)by intervals ranging from minutes to weeks. In embodiments where theother agent and expression construct are applied separately to the cell,one would generally ensure that a significant period of time did notexpire between each delivery, such that the agent and expressionconstruct would still be able to exert an advantageously combined effecton the cell, tissue or subject. In such instances, it is contemplatedthat one would typically contact the cell with both modalities withinabout 12-24 hours of each other and, more preferably, within about 6-12hours of each other, with a delay time of only about 12 hours being mostpreferred. In some situations, it may be desirable to extend the timeperiod for treatment significantly, however, where several days (2, 3,4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse betweenthe respective administrations.

It also is conceivable that more than one administration of either amodulator of lncEGFL7OS, or the other agent will be desired. In thisregard, various combinations may be employed. By way of illustration,where the modulator of lncEGFL7OS is “A” and the other agent is “B,” thefollowing permutations based on 3 and 4 total administrations areexemplary:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/BOther combinations are likewise contemplated.

D. Pharmacological Therapeutic Agents

Pharmacological therapeutic agents and methods of administration,dosages, etc., are well known to those of skill in the art (see forexample, the “Physicians' Desk Reference,” Klaassen's “ThePharmacological Basis of Therapeutics,” “Remington's PharmaceuticalSciences,” and “The Merck Index, Eleventh Edition,” incorporated hereinby reference in relevant parts), and may be combined with the disclosurein light of the disclosures herein. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject, and suchindividual determinations are within the skill of those of ordinaryskill in the art.

Non-limiting examples of a pharmacological therapeutic agent that may beused in the present disclosure include an antihyperlipoproteinemicagent, an antiarteriosclerotic agent, an antithrombotic/fibrinolyticagent, a blood coagulant, an antiarrhythmic agent, an antihypertensiveagent, a vasopressor, a treatment agent for congestive heart failure, anantianginal agent, an antibacterial agent or a combination thereof. Alsocontemplated for combination with an lncEGFL7OS modulator are any of theagents/therapies discussed in Sections IIIA-B, above.

E. Regulation of Therapies

The present disclosure also contemplates methods for scavenging orclearing lncEGFL7OS agonists or antagonists following treatment. Themethod may comprise overexpressing binding sites for the lncEGFL7OSantagonists in target tissues. In another embodiment, the presentdisclosure provides a method for scavenging or clearing lncEGFL7OSfollowing treatment. In one embodiment, the method comprisesoverexpression of binding site regions for lncEGFL7OS in target tissues.The binding site regions preferably contain a binding sequence forlncEGFL7OS. In some embodiments, the binding site may contain a sequencefrom one or more targets of lncEGFL7OS, such as miR-126. In anotherembodiment, a lncEGFL7OS antagonist may be administered after lncEGFL7OSto attenuate or stop the function of the lncRNA. In another embodiment,overexpression or silencing of the lncEGFL7OS regulative genes,including ETS1, ETS2 and MAX, can be used to modulate the function oflncEGFL7OS.

F. Drug Formulations and Routes for Administration to Patients

Where clinical applications are contemplated, pharmaceuticalcompositions will be prepared in a form appropriate for the intendedapplication. Generally, this will entail preparing compositions that areessentially free of pyrogens, as well as other impurities that could beharmful to humans or animals.

One will generally desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present disclosure comprise aneffective amount of the vector or cells, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. The phrases“pharmaceutically acceptable” or “pharmacologically acceptable” refersto molecular entities and compositions that do not produce adverse,allergic, or other untoward reactions when administered to an animal ora human. As used herein, “pharmaceutically acceptable carrier” includessolvents, buffers, solutions, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike acceptable for use in formulating pharmaceuticals, such aspharmaceuticals suitable for administration to humans. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredients of the present disclosure, itsuse in therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions, providedthey do not inactivate the vectors or cells of the compositions.

The active compositions of the present disclosure may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present disclosure may be via any common route so longas the target tissue is available via that route. This includes oral,nasal, or buccal. Alternatively, administration may be by intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection,or by direct injection into cardiac tissue. Such compositions wouldnormally be administered as pharmaceutically acceptable compositions, asdescribed supra.

The active compounds may also be administered parenterally orintraperitoneally. By way of illustration, solutions of the activecompounds as free base or pharmacologically acceptable salts can beprepared in water suitably mixed with a surfactant, such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations generallycontain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include, forexample, sterile aqueous solutions or dispersions and sterile powdersfor the extemporaneous preparation of sterile injectable solutions ordispersions. Generally, these preparations are sterile and fluid to theextent that easy injectability exists. Preparations should be stableunder the conditions of manufacture and storage and should be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi. Appropriate solvents or dispersion media may contain, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the activecompounds in an appropriate amount into a solvent along with any otheringredients (for example as enumerated above) as desired, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the desired otheringredients, e.g., as enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation include vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient(s) plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

For oral administration the polypeptides of the present disclosuregenerally may be incorporated with excipients and used in the form ofnon-ingestible mouthwashes and dentifrices. A mouthwash may be preparedincorporating the active ingredient in the required amount in anappropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan antiseptic wash containing sodium borate, glycerin and potassiumbicarbonate. The active ingredient may also be dispersed in dentifrices,including gels, pastes, powders and slurries. The active ingredient maybe added in a therapeutically effective amount to a paste dentifricethat may include water, binders, abrasives, flavoring agents, foamingagents, and humectants.

The compositions of the present disclosure generally may be formulatedin a neutral or salt form. Pharmaceutically-acceptable salts include,for example, acid addition salts (formed with the free amino groups ofthe protein) derived from inorganic acids (e.g., hydrochloric orphosphoric acids, or from organic acids (e.g., acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups ofthe protein can also be derived from inorganic bases (e.g., sodium,potassium, ammonium, calcium, or ferric hydroxides) or from organicbases (e.g., isopropylamine, trimethylamine, histidine, procaine and thelike.

Upon formulation, solutions are preferably administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations may easily be administeredin a variety of dosage forms such as injectable solutions, drug releasecapsules and the like. For parenteral administration in an aqueoussolution, for example, the solution generally is suitably buffered andthe liquid diluent first rendered isotonic for example with sufficientsaline or glucose. Such aqueous solutions may be used, for example, forintravenous, intramuscular, subcutaneous and intraperitonealadministration. Preferably, sterile aqueous media are employed as isknown to those of skill in the art, particularly in light of the presentdisclosure. By way of illustration, a single dose may be dissolved in 1ml of isotonic NaCl solution and either added to 1000 ml ofhypodermoclysis fluid or injected at the proposed site of infusion, (seefor example, “Remington's Pharmaceutical Sciences” 15th Edition, pages1035-1038 and 1570-1580). Some variation in dosage will necessarilyoccur depending on the condition of the subject being treated. Theperson responsible for administration will, in any event, determine theappropriate dose for the individual subject. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

IV. Kits

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, an individual lncRNA modulator (e.g., lncRNA,expression construct) is included in a kit. The kit may further includewater and hybridization buffer to facilitate hybridization to thelncRNAs. The kit may also include one or more transfection reagent(s) tofacilitate delivery of the lncRNA to cells.

The components of the kits may be packaged either in aqueous media or inlyophilized form. The container means of the kits will generally includeat least one vial, test tube, flask, bottle, syringe or other containermeans, into which a component may be placed, and preferably, suitablyaliquoted. Where there is more than one component in the kit (labelingreagent and label may be packaged together), the kit also will generallycontain a second, third or other additional container into which theadditional components may be separately placed. However, variouscombinations of components may be comprised in a vial. The kits of thepresent disclosure also will typically include a means for containingthe nucleic acids, and any other reagent containers in close confinementfor commercial sale. Such containers may include injection orblow-molded plastic containers into which the desired vials areretained.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred.

However, the components of the kit may be provided as dried powder(s).When reagents and/or components are provided as a dry powder, the powdercan be reconstituted by the addition of a suitable solvent. It isenvisioned that the solvent may also be provided in another containermeans.

The container means will generally include at least one vial, test tube,flask, bottle, syringe and/or other container means, into which thenucleic acid formulations are placed, preferably, suitably allocated.The kits may also comprise a second container means for containing asterile, pharmaceutically acceptable buffer and/or other diluent.

The kits of the present disclosure will also typically include a meansfor containing the vials in close confinement for commercial sale, suchas, e.g., injection and/or blow-molded plastic containers into which thedesired vials are retained.

Such kits may also include components that preserve or maintain thelncRNA or that protect against its degradation. Such components may beRNAse-free or protect against RNAses. Such kits generally will comprise,in suitable means, distinct containers for each individual reagent orsolution.

A kit will also include instructions for employing the kit components aswell the use of any other reagent not included in the kit. Instructionsmay include variations that can be implemented.

It is contemplated that such reagents are embodiments of kits of thedisclosure. Such kits, however, are not limited to the particular itemsidentified above and may include any reagent used for the manipulationor characterization of lncRNA.

V. Screening Methods

The present disclosure further comprises methods for identifyingmodulators of lncEGFL7OS that are useful in the prevention or treatmentof the diseases discussed above. These assays may comprise randomscreening of large libraries of candidate substances; alternatively, theassays may be used to focus on particular classes of compounds selectedwith an eye towards structural attributes that are believed to make themmore likely to modulate the expression and/or function of lncEGFL7OS.

To identify a modulator of lncEGFL7OS, one generally will determine thefunction of a lncEGFL7OS in the presence and absence of the candidatesubstance. For example, a method generally comprises:

-   -   (a) providing a candidate modulator;    -   (b) admixing the candidate modulator with a lncEGFL7OS;    -   (c) measuring lncEGFL7OS activity; and    -   (d) comparing the activity in step (c) with the activity in the        absence of the candidate modulator,        wherein a difference between the measured activities indicates        that the candidate modulator is, indeed, a modulator of        lncEGFL7OS. Assays also may be conducted in isolated cells,        organs, or in living organisms.

It will, of course, be understood that all the screening methods of thepresent disclosure are useful in themselves notwithstanding the factthat effective candidates may not be found. The disclosure providesmethods for screening for such candidates, not solely methods of findingthem.

A. Modulators

As used herein the term “candidate substance” refers to any moleculethat may potentially modulate angiogenic-regulating aspects oflncEGFL7OS. One will typically acquire, from various commercial sources,molecular libraries that are believed to meet the basic criteria foruseful drugs in an effort to “brute force” the identification of usefulcompounds. Screening of such libraries, includingcombinatorially-generated libraries (e.g., compound libraries), is arapid and efficient way to screen large number of related (andunrelated) compounds for activity. Combinatorial approaches also lendthemselves to rapid evolution of potential drugs by the creation ofsecond, third, and fourth generation compounds modeled on active, butotherwise undesirable compounds.

B. In Vitro Assays

A quick, inexpensive and easy assay to run is an in vitro assay. Suchassays generally use isolated molecules, can be run quickly and in largenumbers, thereby increasing the amount of information obtainable in ashort period of time. A variety of vessels may be used to run theassays, including test tubes, plates, dishes and other surfaces such asdipsticks or beads.

A technique for high throughput screening of compounds is described inWO 84/03564. Large numbers of small nucleic acids may be synthesized ona solid substrate, such as plastic pins or some other surface. Suchmolecules can be rapidly screening for their ability to inhibitlncEGFL7OS.

C. In Cyto Assays

The present disclosure also contemplates the screening of compounds fortheir ability to modulate lncEGFL7OS activity and expression in cells.Various cell lines, including those derived from endothelial cells andhematopoietic cells, can be utilized for such screening assays,including cells specifically engineered for this purpose.

D. In Vivo Assays

In vivo assays involve the use of various animal models of vasculardiseases, discussed above, including transgenic animals that have beenengineered to have specific defects, or carry markers that can be usedto measure the ability of a candidate substance to reach and effectdifferent cells within the organism. Due to their size, ease ofhandling, and information on their physiology and genetic make-up, miceare a preferred embodiment, especially for transgenics. However, otheranimals are suitable as well, including rats, rabbits, hamsters, guineapigs, gerbils, woodchucks, cats, dogs, sheep, goats, pigs, cows, horsesand monkeys (including chimps, gibbons and baboons). Assays forinhibitors may be conducted using an animal model derived from any ofthese species.

Treatment of animals with test compounds will involve the administrationof the compound, in an appropriate form, to the animal. Administrationwill be by any route that could be utilized for clinical purposes.Determining the effectiveness of a compound in vivo may involve avariety of different criteria. Also, measuring toxicity and doseresponse can be performed in animals in a more meaningful fashion thanin in vitro or in cyto assays.

VI. Vectors for Cloning, Gene Transfer and Expression

Within certain embodiments expression vectors are employed to expresslncEGFL7OS or related molecules. Expression requires that appropriatesignals be provided in the vectors, and which include various regulatoryelements, such as enhancers/promoters from both viral and mammaliansources that drive expression of the genes of interest in host cells.Elements designed to optimize messenger RNA stability andtranslatability in host cells also are defined. The conditions for theuse of a number of dominant drug selection markers for establishingpermanent, stable cell clones expressing the products are also provided,as is an element that links expression of the drug selection markers toexpression of the polypeptide.

A. Regulatory Elements

Throughout this application, the term “expression construct” is meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid encodingsequence is capable of being transcribed. Generally, the nucleic acidencoding a gene product is under transcriptional control of a promoter.A “promoter” refers to a DNA sequence recognized by the syntheticmachinery of the cell, or introduced synthetic machinery, required toinitiate the specific transcription of a gene. The phrase “undertranscriptional control” means that the promoter is in the correctlocation and orientation in relation to the nucleic acid to control RNApolymerase initiation and expression of the gene.

The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either co-operatively or independently to activatetranscription.

In other embodiments, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter, the Rous sarcoma virus longterminal repeat, rat insulin promoter and glyceraldehyde-3-phosphatedehydrogenase can be used to obtain high-level expression of the codingsequence of interest. The use of other viral or mammalian cellular orbacterial phage promoters which are well-known in the art to achieveexpression of a coding sequence of interest is contemplated as well,provided that the levels of expression are sufficient for a givenpurpose.

By employing a promoter with well-known properties, the level andpattern of expression of the protein of interest following transfectionor transformation can be optimized. Further, selection of a promoterthat is regulated in response to specific physiologic signals can permitinducible expression of the gene product. Tables 1 and 2 list severalregulatory elements that may be employed, in the context of the presentdisclosure, to regulate the expression of the gene of interest. Thislist is not intended to be exhaustive of all the possible elementsinvolved in the promotion of gene expression but, merely, to beexemplary thereof.

Enhancers are genetic elements that increase transcription from apromoter located at a distant position on the same molecule of DNA.Enhancers are organized much like promoters. That is, they are composedof many individual elements, each of which binds to one or moretranscriptional proteins.

The basic distinction between enhancers and promoters is operational. Anenhancer region as a whole must be able to stimulate transcription at adistance; this need not be true of a promoter region or its componentelements. On the other hand, a promoter must have one or more elementsthat direct initiation of RNA synthesis at a particular site and in aparticular orientation, whereas enhancers lack these specificities.Promoters and enhancers are often overlapping and contiguous, oftenseeming to have a very similar modular organization.

Below is a list of viral promoters, cellular promoters/enhancers andinducible promoters/enhancers that could be used in combination with thenucleic acid encoding a gene of interest in an expression construct(Table 1 and Table 2). Additionally, any promoter/enhancer combination(as per the Eukaryotic Promoter Data Base EPDB) could also be used todrive expression of the gene. Eukaryotic cells can support cytoplasmictranscription from certain bacterial promoters if the appropriatebacterial polymerase is provided, either as part of the delivery complexor as an additional genetic expression construct.

TABLE 1 Promoter and/or Enhancer Promoter/Enhancer ReferencesImmunoglobulin Heavy Chain Banerji et al., 1983; Gilles et al., 1983;Grosschedl et al., 1985; Atchinson et al., 1986, 1987; Imler et al.,1987; Weinberger et al., 1984; Kiledjian et al., 1988; Porton et al.;1990 Immunoglobulin Light Chain Queen et al., 1983; Picard et al., 1984T-Cell Receptor Luria et al., 1987; Winoto et al., 1989; Redondo et al.;1990 HLA DQ a and/or DQ β Sullivan et al., 1987 β-Interferon Goodbournet al., 1986; Fujita et al., 1987; Goodbourn et al., 1988 Interleukin-2Greene et al., 1989 Interleukin-2 Receptor Greene et al., 1989; Lin etal., 1990 MHC Class II 5 Koch et al., 1989 MHC Class II HLA-DRa Shermanet al., 1989 β-Actin Kawamoto et al., 1988; Ng et al.; 1989 MuscleCreatine Kinase (MCK) Jaynes et al., 1988; Horlick et al., 1989; Johnsonet al., 1989 Prealbumin (Transthyretin) Costa et al., 1988 Elastase IOmitz et al., 1987 Metallothionein (MTII) Karin et al., 1987; Culotta etal., 1989 Collagenase Pinkert et al., 1987; Angel et al., 1987 AlbuminPinkert et al., 1987; Tronche et al., 1989, 1990 α-Fetoprotein Godboutet al., 1988; Campere et al., 1989 γ-Globin Bodine et al., 1987;Perez-Stable et al., 1990 β-Globin Trudel et al., 1987 c-fos Cohen etal., 1987 c-HA-ras Triesman, 1986; Deschamps et al., 1985 Insulin Edlundet al., 1985 Neural Cell Adhesion Molecule Hirsh et al., 1990 (NCAM)α₁-Antitrypain Latimer et al., 1990 H2B (TH2B) Histone Hwang et al.,1990 Mouse and/or Type I Collagen Ripe et al., 1989 Glucose-RegulatedProteins Chang et al., 1989 (GRP94 and GRP78) Rat Growth Hormone Larsenet al., 1986 Human Serum Amyloid A (SAA) Edbrooke et al., 1989 TroponinI (TN I) Yutzey et al., 1989 Platelet-Derived Growth Factor Pech et al.,1989 (PDGF) Duchenne Muscular Dystrophy Klamut et al., 1990 SV40 Banerjiet al., 1981; Moreau et al., 1981; Sleigh et al., 1985; Firak et al.,1986; Herr et al., 1986; Imbra et al., 1986; Kadesch et al., 1986; Wanget al., 1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner et al.,1988 Polyoma Swartzendruber et al., 1975; Vasseur et al., 1980; Katinkaet al., 1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983; deVilliers et al., 1984; Hen et al., 1986; Satake et al., 1988; Campbelland/or Villarreal, 1988 Retroviruses Kriegler et al., 1982, 1983;Levinson et al., 1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze etal., 1986; Miksicek et al., 1986; Celander et al., 1987; Thiesen et al.,1988; Celander et al., 1988; Choi et al., 1988; Reisman et al., 1989Papilloma Virus Campo et al., 1983; Lusky et al., 1983; Spandidos and/orWilkie, 1983; Spalholz et al., 1985; Lusky et al., 1986; Cripe et al.,1987; Gloss et al., 1987; Hirochika et al., 1987; Stephens et al., 1987Hepatitis B Virus Bulla et al., 1986; Jameel et al., 1986; Shaul et al.,1987; Spandau et al., 1988; Vannice et al., 1988 Human ImmunodeficiencyVirus Muesing et al., 1987; Hauber et al., 1988; Jakobovits et al.,1988; Feng et al., 1988; Takebe et al., 1988; Rosen et al., 1988;Berkhout et al., 1989; Laspia et al., 1989; Sharp et al., 1989; Braddocket al., 1989 CD11b Hickstein et al., 1992 Gibbon Ape Leukemia VirusHolbrook et al., 1987; Quinn et al., 1989

TABLE 2 Inducible Elements Element Inducer References MT II PhorbolEster Palmiter et al., 1982; (TFA) Haslinger et al., 1985; Heavy metalsSearle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987, Karin etal., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTV (mouse mammaryGlucocor- Huang et al., 1981; Lee tumor virus) ticoids et al., 1981;Majors et al., 1983; Chandler et al., 1983; Lee et al., 1984; Ponta etal., 1985; Sakai et al., 1988 β-Interferon poly(rI)x Tavernier et al.,1983 poly(rc) Adenovirus 5 E2 ElA Imperiale et al., 1984 CollagenasePhorbol Ester Angel et al., 1987a (TPA) Stromelysin Phorbol Ester Angelet al., 1987b (TPA) SV40 Phorbol Ester Angel et al., 1987b (TPA) MurineMX Gene Interferon, Hug et al., 1988 Newcastle Disease Virus GRP78 GeneA23187 Resendez et al., 1988 α-2-Macroglobulin IL-6 Kunz et al., 1989Vimentin Serum Rittling et al., 1989 MHC Class I Gene H-2κb InterferonBlanar et al., 1989 HSP70 ElA, SV40 Taylor et al., 1989, 1990a, Large T1990b Antigen Proliferin Phorbol Ester- Mordacq et al., 1989 TPA TumorNecrosis Factor PMA Hensel et al., 1989 Thyroid Stimulating ThyroidChatterjee et al., 1989 Hormone α Gene Hormone

Of particular interest are endothelial cell promoters, such as Tie1,Tie2, Ve-cadherin, EGFL7/lncEGFL7OS or promoters, and muscle specificpromoters, including cardiac specific promoters. These include themyosin light chain-2 promoter (Franz et al., 1994; Kelly et al., 1995),the α-actin promoter (Moss et al., 1996), the troponin 1 promoter(Bhaysar et al., 1996); the Na⁺/Ca²⁺ exchanger promoter (Barnes et al.,1997), the dystrophin promoter (Kimura et al., 1997), the α7 integrinpromoter (Ziober and Kramer, 1996), the brain natriuretic peptidepromoter (LaPointe et al., 1996) and the αB-crystallin/small heat shockprotein promoter (Gopal-Srivastava, 1995), α-myosin heavy chain promoter(Yamauchi-Takihara et al., 1989) and the ANF promoter (LaPointe et al.,1988).

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the disclosure, and any suchsequence may be employed such as human growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression cassette is a terminator. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

B. Selectable Markers

In certain embodiments of the disclosure, the cells contain nucleic acidconstructs of the present disclosure, a cell may be identified in vitroor in vivo by including a marker in the expression construct. Suchmarkers would confer an identifiable change to the cell permitting easyidentification of cells containing the expression construct. Usually theinclusion of a drug selection marker aids in cloning and in theselection of transformants, for example, genes that confer resistance toneomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol areuseful selectable markers. Alternatively, enzymes such as herpes simplexvirus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT)may be employed. Immunologic markers also can be employed. Theselectable marker employed is not believed to be important, so long asit is capable of being expressed simultaneously with the nucleic acidencoding a gene product. Further examples of selectable markers are wellknown to one of skill in the art.

C. Delivery of Expression Vectors

There are a number of ways in which expression vectors may introducedinto cells. In certain embodiments of the disclosure, the expressionconstruct comprises a virus or engineered construct derived from a viralgenome. The ability of certain viruses to enter cells viareceptor-mediated endocytosis, to integrate into host cell genome andexpress viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign genes into mammalian cells(Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden,1986; Temin, 1986). The first viruses used as gene vectors were DNAviruses including the papovaviruses (simian virus 40, bovine papillomavirus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) andadenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have arelatively low capacity for foreign DNA sequences and have a restrictedhost spectrum. Furthermore, their oncogenic potential and cytopathiceffects in permissive cells raise safety concerns. They can accommodateonly up to 8 kB of foreign genetic material but can be readilyintroduced in a variety of cell lines and laboratory animals (Nicolasand Rubinstein, 1988; Temin, 1986).

One of the preferred methods for in vivo delivery involves the use of anadenovirus expression vector. “Adenovirus expression vector” is meant toinclude those constructs containing adenovirus sequences sufficient to(a) support packaging of the construct and (b) to express an antisensepolynucleotide that has been cloned therein. In this context, expressiondoes not require that the gene product be synthesized.

The expression vector comprises a genetically engineered form ofadenovirus. Knowledge of the genetic organization of adenovirus, a 36kB, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kB (Grunhaus andHorwitz, 1992). In contrast to retrovirus, the adenoviral infection ofhost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner without potential genotoxicity.Also, adenoviruses are structurally stable, and no genome rearrangementhas been detected after extensive amplification. Adenovirus can infectvirtually all epithelial cells regardless of their cell cycle stage. Sofar, adenoviral infection appears to be linked only to mild disease suchas acute respiratory disease in humans.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget cell range and high infectivity. Both ends of the viral genomecontain 100-200 base pair inverted repeats (ITRs), which are ciselements necessary for viral DNA replication and packaging. The early(E) and late (L) regions of the genome contain different transcriptionunits that are divided by the onset of viral DNA replication. The E1region (E1A and E1B) encodes proteins responsible for the regulation oftranscription of the viral genome and a few cellular genes. Theexpression of the E2 region (E2A and E2B) results in the synthesis ofthe proteins for viral DNA replication. These proteins are involved inDNA replication, late gene expression and host cell shut-off (Renan,1990). The products of the late genes, including the majority of theviral capsid proteins, are expressed only after significant processingof a single primary transcript issued by the major late promoter (MLP).The MLP, (located at 16.8 m.u.) is particularly efficient during thelate phase of infection, and all the mRNA's issued from this promoterpossess a 5′-tripartite leader (TPL) sequence which makes them preferredmRNA's for translation.

In a current system, recombinant adenovirus is generated from homologousrecombination between shuttle vector and provirus vector. Due to thepossible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

Generation and propagation of the current adenovirus vectors, which arereplication deficient, depend on a unique helper cell line, designated293, which was transformed from human embryonic kidney cells by Ad5 DNAfragments and constitutively expresses E1 proteins (Graham et al.,1977). Since the E3 region is dispensable from the adenovirus genome(Jones and Shenk, 1978), the current adenovirus vectors, with the helpof 293 cells, carry foreign DNA in either the E1, the D3 or both regions(Graham and Prevec, 1991). In nature, adenovirus can packageapproximately 105% of the wild-type genome (Ghosh-Choudhury et al.,1987), providing capacity for about 2 extra kb of DNA. Combined with theapproximately 5.5 kb of DNA that is replaceable in the E1 and E3regions, the maximum capacity of the current adenovirus vector is under7.5 kb, or about 15% of the total length of the vector. More than 80% ofthe adenovirus viral genome remains in the vector backbone and is thesource of vector-borne cytotoxicity. Also, the replication deficiency ofthe E1-deleted virus is incomplete.

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the preferred helper cell line is 293.

Racher et al. (1995) disclosed improved methods for culturing 293 cellsand propagating adenovirus. In one format, natural cell aggregates aregrown by inoculating individual cells into 1 liter siliconized spinnerflasks (Techne, Cambridge, UK) containing 100-200 ml of medium.Following stirring at 40 rpm, the cell viability is estimated withtrypan blue. In another format, Fibra-Cel microcarriers (Bibby Sterlin,Stone, UK) (5 g/l) is employed as follows. A cell inoculum, resuspendedin 5 ml of medium, is added to the carrier (50 ml) in a 250 mlErlenmeyer flask and left stationary, with occasional agitation, for 1to 4 h. The medium is then replaced with 50 ml of fresh medium andshaking initiated. For virus production, cells are allowed to grow toabout 80% confluence, after which time the medium is replaced (to 25% ofthe final volume) and adenovirus added at an MOI of 0.05. Cultures areleft stationary overnight, following which the volume is increased to100% and shaking commenced for another 72 h.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the disclosure. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in thepresent disclosure. This is because Adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

As stated above, the typical vector according to the present disclosureis replication defective and will not have an adenovirus E1 region.Thus, it will be most convenient to introduce the polynucleotideencoding the gene of interest at the position from which the E1-codingsequences have been removed. However, the position of insertion of theconstruct within the adenovirus sequences is not critical to thedisclosure. The polynucleotide encoding the gene of interest may also beinserted in lieu of the deleted E3 region in E3 replacement vectors, asdescribed by Karlsson et al. (1986), or in the E4 region where a helpercell line or helper virus complements the E4 defect.

Adenovirus is easy to grow and manipulate and exhibits broad host rangein vitro and in vivo. This group of viruses can be obtained in hightiters, e.g., 10⁹-10¹² plaque-forming units per ml, and they are highlyinfective. The life cycle of adenovirus does not require integrationinto the host cell genome. The foreign genes delivered by adenovirusvectors are episomal and, therefore, have low genotoxicity to hostcells. No side effects have been reported in studies of vaccination withwild-type adenovirus (Couch et al., 1963; Top et al., 1971),demonstrating their safety and therapeutic potential as in vivo genetransfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhausand Horwitz, 1992; Graham and Prevec, 1991). Recently, animal studiessuggested that recombinant adenovirus could be used for gene therapy(Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet etal., 1990; Rich et al., 1993). Studies in administering recombinantadenovirus to different tissues include trachea instillation (Rosenfeldet al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al.,1993), peripheral intravenous injections (Herz and Gerard, 1993) andstereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol, and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene contains a signal for packaging of the genome into virions. Twolong terminal repeat (LTR) sequences are present at the 5′ and 3′ endsof the viral genome. These contain strong promoter and enhancersequences and are also required for integration in the host cell genome(Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding agene of interest is inserted into the viral genome in the place ofcertain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into this cell line (by calciumphosphate precipitation for example), the packaging sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubinstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is then collected, optionally concentrated,and used for gene transfer. Retroviral vectors are able to infect abroad variety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al., 1975).

A novel approach designed to allow specific targeting of retrovirusvectors was recently developed based on the chemical modification of aretrovirus by the chemical addition of lactose residues to the viralenvelope. This modification could permit the specific infection ofhepatocytes via sialoglycoprotein receptors.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, they demonstrated the infection of avariety of human cells that bore those surface antigens with anecotropic virus in vitro (Roux et al., 1989).

There are certain limitations to the use of retrovirus vectors in allaspects of the present disclosure. For example, retrovirus vectorsusually integrate into random sites in the cell genome. This can lead toinsertional mutagenesis through the interruption of host genes orthrough the insertion of viral regulatory sequences that can interferewith the function of flanking genes (Varmus et al., 1981). Anotherconcern with the use of defective retrovirus vectors is the potentialappearance of wild-type replication-competent virus in the packagingcells. This can result from recombination events in which theintact-sequence from the recombinant virus inserts upstream from thegag, pol, env sequence integrated in the host cell genome. However, newpackaging cell lines are now available that should greatly decrease thelikelihood of recombination (Markowitz et al., 1988; Hersdorffer et al.,1990).

Other viral vectors may be employed as expression constructs in thepresent disclosure. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988)adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986;Hermonat and Muzycska, 1984) and herpesviruses may be employed. Theyoffer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988; Horwich et al., 1990).

With the recognition of defective hepatitis B viruses, new insight wasgained into the structure-function relationship of different viralsequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. The hepatotropism and persistence(integration) were particularly attractive properties for liver-directedgene transfer. Chang et al., introduced the chloramphenicolacetyltransferase (CAT) gene into duck hepatitis B virus genome in theplace of the polymerase, surface, and pre-surface coding sequences. Itwas co-transfected with wild-type virus into an avian hepatoma cellline. Culture media containing high titers of the recombinant virus wereused to infect primary duckling hepatocytes. Stable CAT gene expressionwas detected for at least 24 days after transfection (Chang et al.,1991).

In order to effect expression of sense or antisense gene constructs, theexpression construct must be delivered into a cell. This delivery may beaccomplished in vitro, as in laboratory procedures for transformingcells lines, or in vivo or ex vivo, as in the treatment of certaindisease states. One mechanism for delivery is via viral infection wherethe expression construct is encapsidated in an infectious viralparticle.

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells also are contemplated by the presentdisclosure. These include calcium phosphate precipitation (Graham andVan Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990)DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al., 1986;Potter et al., 1984), direct microinjection (Harland and Weintraub,1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al.,1979) and lipofectamine-DNA complexes, cell sonication (Fechheimer etal., 1987), gene bombardment using high velocity microprojectiles (Yanget al., 1990), and receptor-mediated transfection (Wu and Wu, 1987; Wuand Wu, 1988). Some of these techniques may be successfully adapted forin vivo or ex vivo use.

Once the expression construct has been delivered into the cell thenucleic acid encoding the gene of interest may be positioned andexpressed at different sites. In certain embodiments, the nucleic acidencoding the gene may be stably integrated into the genome of the cell.This integration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle. How the expression construct is delivered to a cell and where inthe cell the nucleic acid remains is dependent on the type of expressionconstruct employed.

In yet another embodiment of the disclosure, the expression constructmay simply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above whichphysically or chemically permeabilize the cell membrane. This isparticularly applicable for transfer in vitro but it may be applied toin vivo use as well. Dubensky et al. (1984) successfully injectedpolyomavirus DNA in the form of calcium phosphate precipitates intoliver and spleen of adult and newborn mice demonstrating active viralreplication and acute infection. Benvenisty and Neshif (1986) alsodemonstrated that direct intraperitoneal injection of calciumphosphate-precipitated plasmids results in expression of the transfectedgenes. It is envisioned that DNA encoding a gene of interest may also betransferred in a similar manner in vivo and express the gene product.

In still another embodiment of the disclosure for transferring a nakedDNA expression construct into cells may involve particle bombardment.This method depends on the ability to accelerate DNA-coatedmicroprojectiles to a high velocity allowing them to pierce cellmembranes and enter cells without killing them (Klein et al., 1987).Several devices for accelerating small particles have been developed.One such device relies on a high voltage discharge to generate anelectrical current, which in turn provides the motive force (Yang etal., 1990). The microprojectiles used have consisted of biologicallyinert substances such as tungsten or gold beads.

Selected organs including the liver, skin, and muscle tissue of rats andmice have been bombarded in vivo (Yang et al., 1990; Zelenin et al.,1991). This may require surgical exposure of the tissue or cells, toeliminate any intervening tissue between the gun and the target organ,i.e., ex vivo treatment. Again, DNA encoding a particular gene may bedelivered via this method and still be incorporated by the presentdisclosure.

In a further embodiment of the disclosure, the expression construct maybe entrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful. Wong et al., (1980) demonstrated thefeasibility of liposome-mediated delivery and expression of foreign DNAin cultured chick embryo, HeLa and hepatoma cells. Nicolau et al.,(1987) accomplished successful liposome-mediated gene transfer in ratsafter intravenous injection.

In certain embodiments of the disclosure, the liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the liposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression constructshave been successfully employed in transfer and expression of nucleicacid in vitro and in vivo, then they are applicable for the presentdisclosure. Where a bacterial promoter is employed in the DNA construct,it also will be desirable to include within the liposome an appropriatebacterial polymerase.

Other expression constructs which can be employed to deliver a nucleicacid encoding a particular gene into cells are receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis in almost all eukaryoticcells. Because of the cell type-specific distribution of variousreceptors, the delivery can be highly specific (Wu and Wu, 1993).

Receptor-mediated gene targeting vehicles generally consist of twocomponents: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and transferrin (Wagner et al., 1990). Recently, asynthetic neoglycoprotein, which recognizes the same receptor as ASOR,has been used as a gene delivery vehicle (Ferkol et al., 1993; Peraleset al., 1994) and epidermal growth factor (EGF) has also been used todeliver genes to squamous carcinoma cells (Myers, EPO 0273085).

In other embodiments, the delivery vehicle may comprise a ligand and aliposome. For example, Nicolau et al. (1987) employed lactosyl-ceramide,a galactose-terminal asialganglioside, incorporated into liposomes andobserved an increase in the uptake of the insulin gene by hepatocytes.Thus, it is feasible that a nucleic acid encoding a particular gene alsomay be specifically delivered into a cell type by any number ofreceptor-ligand systems with or without liposomes. For example,epidermal growth factor (EGF) may be used as the receptor for mediateddelivery of a nucleic acid into cells that exhibit upregulation of EGFreceptor. Mannose can be used to target the mannose receptor on livercells. Also, antibodies to CD5 (CLL), CD22 (lymphoma), CD25 (T-cellleukemia) and MAA (melanoma) can similarly be used as targetingmoieties.

In a particular example, the oligonucleotide may be administered incombination with a cationic lipid. Examples of cationic lipids include,but are not limited to, lipofectin, DOTMA, DOPE, and DOTAP. Thepublication of WO/0071096, which is specifically incorporated byreference, describes different formulations, such as a DOTAP:cholesterolor cholesterol derivative formulation that can effectively be used forgene therapy. Other disclosures also discuss different lipid orliposomal formulations including nanoparticles and methods ofadministration; these include, but are not limited to, U.S. PatentPublication 20030203865, 20020150626, 20030032615, and 20040048787,which are specifically incorporated by reference to the extent theydisclose formulations and other related aspects of administration anddelivery of nucleic acids. Methods used for forming particles are alsodisclosed in U.S. Pat. Nos. 5,844,107, 5,877,302, 6,008,336, 6,077,835,5,972,901, 6,200,801, and 5,972,900, which are incorporated by referencefor those aspects.

In certain embodiments, gene transfer may more easily be performed underex vivo conditions. Ex vivo gene therapy refers to the isolation ofcells from an animal, the delivery of a nucleic acid into the cells invitro, and then the return of the modified cells back into an animal.This may involve the surgical removal of tissue/organs from an animal orthe primary culture of cells and tissues.

VII. Methods of Making Transgenic Mice

A particular embodiment of the present disclosure provides transgenicanimals that lack one or both functional lncEGFL7OS alleles. Also,transgenic animals that express lncEGFL7OS under the control of aninducible, tissue selective or a constitutive promoter, recombinant celllines derived from such animals, are contemplated. The use of aninducible or repressable lncEGFL7OS encoding nucleic acid provides amodel for over- or unregulated expression. Also, transgenic animals thatare “knocked out” for lncEGFL7OS, in one or both alleles, arecontemplated. Also, transgenic animals that are “knocked out” forlncEGFL7OS, in one or both alleles for one or both clusters, arecontemplated.

In a general aspect, a transgenic animal is produced by the integrationof a given transgene into the genome in a manner that permits theexpression of the transgene. Methods for producing transgenic animalsare generally described by Wagner and Hoppe (U.S. Pat. No. 4,873,191;incorporated herein by reference), and Brinster et al. (1985;incorporated herein by reference).

Typically, a gene flanked by genomic sequences is transferred bymicroinjection into a fertilized egg. The microinjected eggs areimplanted into a host female, and the progeny are screened for theexpression of the transgene. Transgenic animals may be produced from thefertilized eggs from a number of animals including, but not limited toreptiles, amphibians, birds, mammals, and fish.

DNA clones for microinjection can be prepared by any means known in theart. For example, DNA clones for microinjection can be cleaved withenzymes appropriate for removing the bacterial plasmid sequences, andthe DNA fragments electrophoresed on 1% agarose gels in TBE buffer,using standard techniques. The DNA bands are visualized by staining withethidium bromide, and the band containing the expression sequences isexcised. The excised band is then placed in dialysis bags containing 0.3M sodium acetate, pH 7.0. DNA is electroeluted into the dialysis bags,extracted with a 1:1 phenol:chloroform solution and precipitated by twovolumes of ethanol. The DNA is redissolved in 1 ml of low salt buffer(0.2 M NaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) and purified on anElutip-D™ column. The column is first primed with 3 ml of high saltbuffer (1 M NaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) followed by washingwith 5 ml of low salt buffer. The DNA solutions are passed through thecolumn three times to bind DNA to the column matrix. After one wash with3 ml of low salt buffer, the DNA is eluted with 0.4 ml high salt bufferand precipitated by two volumes of ethanol. DNA concentrations aremeasured by absorption at 260 nm in a UV spectrophotometer. Formicroinjection, DNA concentrations are adjusted to 3 μg/ml in 5 mM Tris,pH 7.4 and 0.1 mM EDTA. Other methods for purification of DNA formicroinjection are described in in Palmiter et al. (1982); and inSambrook et al. (2001).

In an exemplary microinjection procedure, female mice six weeks of ageare induced to superovulate with a 5 IU injection (0.1 cc, ip) ofpregnant mare serum gonadotropin (PMSG; Sigma) followed 48 hours laterby a 5 IU injection (0.1 cc, ip) of human chorionic gonadotropin (hCG;Sigma). Females are placed with males immediately after hCG injection.Twenty-one hours after hCG injection, the mated females are sacrificedby C02 asphyxiation or cervical dislocation and embryos are recoveredfrom excised oviducts and placed in Dulbecco's phosphate buffered salinewith 0.5% bovine serum albumin (BSA; Sigma). Surrounding cumulus cellsare removed with hyaluronidase (1 mg/ml). Pronuclear embryos are thenwashed and placed in Earle's balanced salt solution containing 0.5% BSA(EBSS) in a 37.5° C. incubator with a humidified atmosphere at 5% CO₂,95% air until the time of injection. Embryos can be implanted at thetwo-cell stage.

Randomly cycling adult female mice are paired with vasectomized males.C57BL/6 or Swiss mice or other comparable strains can be used for thispurpose. Recipient females are mated at the same time as donor females.At the time of embryo transfer, the recipient females are anesthetizedwith an intraperitoneal injection of 0.015 ml of 2.5% avertin per gramof body weight. The oviducts are exposed by a single midline dorsalincision. An incision is then made through the body wall directly overthe oviduct. The ovarian bursa is then torn with watchmaker's forceps.Embryos to be transferred are placed in DPBS (Dulbecco's phosphatebuffered saline) and in the tip of a transfer pipet (about 10 to 12embryos). The pipet tip is inserted into the infundibulum and theembryos transferred. After the transfer, the incision is closed by twosutures.

VIII. Definitions

The term “treatment” or grammatical equivalents encompasses theimprovement and/or reversal of the symptoms of disease. “Improvement inthe physiologic function” of the heart may be assessed using any of themeasurements described herein, as well as any effect upon the animal'ssurvival. In use of animal models, the response of treated transgenicanimals and untreated transgenic animals is compared using any of theassays described herein (in addition, treated and untreatednon-transgenic animals may be included as controls).

The term “compound” refers to any chemical entity, pharmaceutical, drug,and the like that can be used to treat or prevent a disease, illness,sickness, or disorder of bodily function. Compounds comprise both knownand potential therapeutic compounds. A compound can be determined to betherapeutic by screening using the screening methods of the presentdisclosure. A “known therapeutic compound” refers to a therapeuticcompound that has been shown (e.g., through animal trials or priorexperience with administration to humans) to be effective in suchtreatment. In other words, a known therapeutic compound is not limitedto a compound efficacious in the treatment of heart failure.

As used herein, the term “agonist” refers to molecules or compounds thatmimic or promote the action of a “native” or “natural” compound.Agonists may be homologous to these natural compounds in respect toconformation, charge or other characteristics. Agonists may includeproteins, nucleic acids, carbohydrates, small molecule pharmaceuticalsor any other molecules that interact with a molecule, receptor, and/orpathway of interest.

As used herein, the terms “antagonist” and “inhibitor” refer tomolecules, compounds, or nucleic acids that inhibit the action of afactor. Antagonists may or may not be homologous to these naturalcompounds in respect to conformation, charge or other characteristics.Antagonists may have allosteric effects that prevent the action of anagonist. Alternatively, antagonists may prevent the function of theagonist. Antagonists and inhibitors may include proteins, nucleic acids,carbohydrates, small molecule pharmaceuticals or any other moleculesthat bind or interact with a receptor, molecule, and/or pathway ofinterest.

As used herein, the term “modulate” refers to a change or an alterationin a biological activity. Modulation may be an increase or a decrease inprotein activity, a change in kinase activity, a change in bindingcharacteristics, or any other change in the biological, functional, orimmunological properties associated with the activity of a protein orother structure of interest. The term “modulator” refers to any moleculeor compound which is capable of changing or altering biological activityas described above.

IX. Examples

The following examples are included to demonstrate preferredembodiments. It should be appreciated by those of skill in the art thatthe techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof embodiments, and thus can be considered to constitute preferred modesfor its practice. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of thedisclosure.

Example 1—Materials and Methods

Animals and In Vivo Angiogenesis Assay.

Animal studies were conducted in accordance with the ARVO statement forthe Use of Animals in Ophthalmic and Vision Research and were approvedby the Institutional Animal Care and Use Committees at the TulaneUniversity. BALB/cAnN-nu (Nude) mice (6 to 8 weeks of age) from Jacksonlab were used for in vivo angiogenesis assay. In vivo Matrigel analysiswas performed. HUVEC cells transfected with control si-RNA, or mix ofsi-LncEGFL7OS#1 and si-LncEGFL7OS#2 (50 nM each) for 2 days. Cells werethen trypsinized and about 5×10⁵ cells were mixed with 50 μl EBM-2 and350 μl ice-cold Matrigel (BD Biosciences). The mixture was then appliedunder the back skin of 8 week-old BALB/cAnN-nu (Nude) female mice. After14 days, The Matrigel plugs were extracted and snap-frozen in OCT andprocessed for immunostaining with human EC marker PECAM-1 and tubelength quantification.

Cell Culture.

HUVEC (ATCC) cells were grown in EC growth medium EGM-2 (Lonza). HCECand HREC cells were kindly provided by Dr. Ashwath Jayagapol fromVanderbilt University and grown in EGM2 media (Lonza). ARPE-19 (ATCC)cells were growth in DMEM/F12 (HyClone) media 20 with 10% FBS. HDF(ATCC) cells were grown in DMEM (HyClone) with 10% FBS. For VEGFtreatment, HUVECs were starved with EC basal medium-2 with 0.1% FBS for24 h and then treated with VEGF (20 ng/mL) for the indicated periods oftime. SiRNA transfection in cell culture was performed as described(Zhou et al., 2013). SiRNAs for LncEGFL7OS were purchased from fromsigma. Sequences for siRNAs are as follows: si-lncEGFL7OS#1:5′-GCGUUUCCCUAGCAAUGUUdTdT-3′ (SEQ ID NO: 2) and5′-AACAUUGCUAGGGAAACGCdTdT-3′ (SEQ ID NO: 3); silncEGFL7OS#2:5′-CAGCUUUGCCCUAUCCCAUdTdT-3′ (SEQ ID NO: 4) and5′-AUGGGAUAGGGCAAAGCUGdTdT-3′ (SEQ ID NO: 5).

LncRNA Microarray.

RNAs from five cell lines were purified by mirVana™ total RNA IsolationKit (Ambion, Invitrogen). These RNAs were subjected to microarray-basedglobal transcriptome analysis (Arraystar Human LncRNA array (version2.0), Arraystar Inc, Rockville, Md.). This LncRNA microarray is designedto detect about 30,586 LncRNAs and 26,109 coding transcripts. TheLncRNAs were constructed using the most highly-respected publictranscriptome databases (Refseq, UCSC known genes, Gencode, etc.), aswell as landmark publications. The lncRNA probes include 19590intergenic lncRNAs (lincRNAs), 4409 intronic lncRNAs, 1299 bidirectionallncRNAs, 1597 sense overlapping lncRNAs and 3691 antisense lncRNAs. Dataanalyses, including hierarchy clustering analysis and functionalenrichment analysis, were performed using Genescript software.

Cell Proliferation, Cell Cycle Analysis, TUNEL Assay Scratch-Wound, InVitro Matrigel Assays.

EC cell proliferation, TUNEL assay and scratch-wound assays wereperformed using HUVEC cells. For cell proliferation assay, about 2×10³transfected HUVECs were seeded in 96-well plates. After starvation with0.1% serum for overnight, the cells were stimulated with 20 ng/mL VEGF-Afor 20 hours and then subjected to BrDU labeling for 4 hours. DNAsynthesis as determined by BrDU incorporation was quantified using acommercial ELISA kit from Roche according to the manufacturer'sinstructions. Cell cycle analysis was performed using Guava® Cell CycleReagents (Guava Technologies) on a Guava instrument and analyzed usingCytosoft™ software according to the manufactural manual. For scratchwound assay, scratchwound was made using a 200-μ1 pipette tip in lncRNAor control siRNA-transfected HUVEC monolayer before VEGF (20 ng/ml)stimulation. 1 μM of 5-fluouracil (Sigma) was then added to the cellsright after scratch wound to block cell proliferation. Post-scratch ECmigration was scored at 14 h after wound scratch. For in vitroangiogenesis assay, at 3 days after lncRNA or control siRNA transfectionwith Liptofectamine RNAiMAX™ reagent (Invitrogen), cells were harvestedfor RNA or in vitro Matrigel assay and branch point analysis asdescribed before.

In Vitro EC-Fibroblast Co-Culture Angiogenesis Assay.

In vitro EC-Fibroblast co-culture was performed. Briefly, HDF wereseeded into each well of a 12-well plate and maintained in DMEM withmedium changes every 2-3 days until they developed confluent monolayers.HUVECs were maintained as described above and transfected with siRNA oneday prior to seeding on HDF monolayers. Approximately 3×10⁴ HUVECs wereseeded onto each monolayer and the HDF/HUVEC co-culture was maintainedfor 7-14 days in EGM-2 medium with medium changes every 2-3 days toallow endothelial cell polarization, migration, networking, and theformation of an in vitro primitive vascular plexus. After 7-14 days thewells were fixed with 100% Methanol at −20° C. for 20 minutes and thenstained with anti-VWF antibody (Dako). After hybridizing a secondaryantibody, the endothelial tissue was visualized and imaged under a Nikonmicroscope. Multiple images were automatically stitched with Nikonsoftware to provide a large image (several mm²) and the resulting imagewas analyzed on ImageJ software to determine the degree ofvascularization. Three wells were used for each condition and resultsare representative of the mean of each three well group.

Ex Vivo Human Choroid Sprouting Assay.

Ex vivo human choroid sprouting assay was adapted from a mouse protocol.Donated human eyeballs were obtained from Southern eye bank (NewOrleans, La.). Eyes were cleaned and kept in sterile ice-cold PBS withPenicillin/Streptomycin before dissection. Using fine forceps, thecornea and the lens from the anterior of the eye were removed. Theperipheral choroid-scleral complex was separated from the retina and theRPE layer was peeled away using fine forceps. The choroid-scleralcomplex was then cut into approximately 1 mm×1 mm pieces using sterilescalpel blade under laminar airflow. The choroid was then washed withsterile ice-cold PBS and transferred into endothelial base medium (EBM2)with 0.1% FBS (300 μl/well in 24-well plates).

The choroid was transfected with control si-RNA, or mix ofsi-LncEGFL7OS#1 and si-LncEGFL7OS#2 (50 nM each) for overnight. Choroidfragments were then washed by EGM2 media then placed in growthfactor-reduced Matrigel™ (BD Biosciences) in 24-well plate. Briefly, 30μl of matrigel was used to coat the bottom of 24 well plates withouttouching the edge of the well. After seeding the choroid, the plate wasincubated in a 37° C. cell culture incubator to make the Matrigelsolidify. 500 μl EC growth medium (EGM-2 with 3% of mouse serum) wereadded slowly to the plate without disturbing the Matrigel, and the platewas incubated at 37° C. cell culture incubator with 5% CO₂. Cell culturemedium was changed every 48 hours. The EC sprouts normally start toappear on the day 5 and grow rapidly between day 8 and 10. Phasecontrast photos of individual explants were taken using a Nikonmicroscope. The sprouting distance was quantified with computer softwareImageJ (National Institute of Health). Sprouting ECs were stained withICAM-2 and isolectin B4.

RNA, Western Blot Analysis and Immunofluorescence.

Human total RNA master panel II was purchased from Clontech (Takara).Total RNA was isolated from human choroid tissues or cell lines usingTRIzol reagent (Invitrogen). Real-time qRT-PCR was performed usingiScript™ cDNA Synthesis system (BioRad), miRNA Real-time qRT-PCR wasperformed using gScript™ cDNA Synthesis and microRNA QuantificationSystem (Quanta Biosciences). Primers for real-time PCRs include humanβ-actin, 5′-0 GAGCAAGAGATGGCCACGG-3′ (SEQ ID NO: 6) and5′-ACTCCATGCCCAGGAAGGAA-3′ (SEQ ID NO: 7); lnc-FLI1-AS1, up:5′-CCTGAGGCCATCTTACCACC-3′ (SEQ ID NO: 8), down:5′-AATCCGCTTCGATGAGTGGG-3′ (SEQ ID NO: 9); lnc-GATA2-AS, up:5′-CGGGCAGCTTACGATTCTTC-3′ (SEQ ID NO: 10), down:5′-CGGTGTCTTTCAGAGGGTCT-3′ (SEQ ID NO: 11); lnc-ECE1, up:5′-CCATGTCGCCTCAGCCTAAA-3′ (SEQ ID NO: 12), down:5′-GGGCAGTCTCAGGGTAACAC-3′ (SEQ ID NO: 13); lnc-ESAM, up:5′-CTCGGAAAACGGAGGGTTGA-3′ (SEQ ID NO: 14), down:5′-CGCTGCCCTTAATTCCTTGC-3′ (SEQ ID NO: 15); lnc-ROBO4-AS, up:5′-ACCAGCAGACCCTGAAACTC-3′ (SEQ ID NO: 16), down:5′-GGCAGGGATCAGGCATTCAT-3′ (SEQ ID NO: 17); lnc-EGFL7-AS, up:5-AGTGCCAGCTTTGCCCTATC-3′ (SEQ ID NO: 18), down:5′-GAGAACACAGGACGTCCACA-3′ (SEQ ID NO: 19); EGFL7-A, up:5-CTTCAGAGGCCAAAAGCACC-3′ (SEQ ID NO: 20), down:5′-GAATCAGTCATCCCCCGGAC-3′ (SEQ ID NO: 21); 20 EGFL7-B, up:5-AAGGGAGGCTCCTGTGGA-3′ (SEQ ID NO: 22), down: 5′-CCTGGGGGCTGCTGATG-3′(SEQ ID NO: 23); EGFL7-C, up: 5-CGGATCCGGCGGCCA-3′ (SEQ ID NO: 24),down: 5′-CGAACGACTCGGAGACAGG-3′ (SEQ ID NO: 25). For Western blotanalysis, protein lysates were resolved by SDS-PAGE and blotted usingstandard procedures. Antibodies used were as follows: ERK1/2 (Cellsignaling), PhosphoPage ERK1/2 (Cell signaling), AKT (Cell signaling),Phospho-AKT (Cell signaling), EGFL-7 (Abacm) and β-Tublin (Abeam). Forimmunofluorescence experiments, samples were fixed with 4%paraformaldehyde or methanol for 30 min. After treatment with 1% TritonX-100 in PBS, samples were incubated in PBS containing 4% goat serum for30 min. The samples were then incubated with primary antibodiesovernight at 4° C., followed by incubation with appropriate secondaryantibodies. Antibody used for immunofluorescence include: vWF (DAKO),ICAM2 (BD Pharmingen), PECAM-1 (DAKO).

Statistics.

Each experiment was repeated at least three times. Student's t-testswere used to determine statistically significant differences betweengroups. P-values of less than 0.05 were considered to be statisticallysignificant.

Example 2—Results

Microarray profiling of lncRNAs in ECs. To identify lncRNAs enriched inECs, a microarray was performed to profile ˜30,000 lncRNAs and ˜26,000coding transcripts using an Arraystar human LncRNA microarray v3.0system (Arraystar, Rockville, Md.). Three primary human EC lines and twonon-EC lines at low passages, namely, human umbilical vein EC (HUVEC),human retinal EC (HREC), human choroidal EC, human dermal fibroblastcell (HDF) and human retinal pigment epithelial (RPE) cell lines, wereused in the array. Purity of EC lines was confirmed by acetyl-LDL uptakeand EC marker staining (FIG. 8). Hierarchical cluster analysis of thearray results validated the clustering of EC lines, which clearlyseparates from the HDF and RPE cell lines based on lncRNA and mRNAexpression (FIG. 1A). Moreover, lncRNAs appeared to be a strongerclassifier to distinguish between EC and non-ECs than mRNAs. 498 lncRNAsare enriched in all three EC lines for more than 2 folds compared to thenon-ECs (see FIG. 1B for top 50 hits). Among them, 308 are intergeniclncRNAs, 62 are sense overlapping lncRNAs, 83 are antisense lncRNAs, 23are bidirectional lncRNAs, and 22 lncRNAs were previously identified aspseudogenes (FIG. 1C). When these lncRNAs were cross-referenced with theenhancer-like lncRNAs, 19 of them are known enhancer-like lncRNAs withnearby coding genes within 300 kB (Orom et al., 2011). the inventorsalso took advantage of the inventors' microarray system in profilingboth lncRNAs and mRNAs, and examined the lncRNA/mRNA regulationrelationship for the EC-enriched lncRNAs. Since many lncRNAs have beenshown to exert locus specific effect on nearby genes, the inventorsfirst performed a bioinformatics search for protein-coding genes thatare within 10 kB of the 498 EC-enriched lncRNAs. 91 lncRNAs haveprotein-coding genes within 10 kB of the lncRNA gene. Moreover, 27 ofthe 91 lncRNAs exhibited parallel expression pattern to the neighboringmRNAs in all 5 cell lines tested, while three of them showed inverseexpression pattern relationship with the neighboring mRNAs. For somelncRNAs, including those near to SRGN, FOXC2, STEAP1B, ECE1, GOT2, EGFL7and PRKAR1B, the specificity for lncRNA in ECs is more robust than theneighboring mRNAs; for some other lncRNAs, including those near to HHIP,ESAM, and UBE2L3, their EC-specificity is less robust than theirneighboring mRNAs.

These results suggest that lncRNA can serve as robust EC-enriched geneexpression markers. The inventors also carried out a functionalenrichment analysis based on the EC-enriched lncRNAs and the associatedgenes. The following biological processes and genes are highlyrepresented in the associated lncRNAs with a false discovery rate (FDR)of less than 10% (FIG. 1D): (1) heart development (NRP1, ECE1, FOXC2,PKD1, ZFPM2, FKBP1A, FOXP4); (2) chordate embryonic development (GATA2,SATB2, ECE1, LMX1B, FOXC2, PKD1, ZFPM2); (3) embryonic developmentending in birth (GATA2, SATB2, ECE1, LMX1B, FOXC2, PKD1, ZFPM2); (4)blood vessel development (NRP1, EGFL7, ROBO4, FOXC2, PKD1, ZFPM2); (5)vasculature development (NRP1, EGFL7, ROBO4, FOXC2, PKD1, ZFPM2); and(6) metallopeptidase activity (ECE1, ADAMTS16, LTA4H, MMP25, ADAM15).From above, genes involved in embryonic development, especially vasculardevelopment, are associated with the EC-enriched lncRNAs. Takentogether, the inventors have established the lncRNA expression profilein ECs by comparative lncRNA microarray, and identified hundreds ofEC-enriched lncRNAs, with a list of them having associated genesinvolved in vascular development.

Confirmation of the EC-Enriched lncRNAs.

Real-time RT-PCR was used to confirm a selected list of EC-enrichedlncRNAs from the microarray. Friend leukemia integration 1 (FLI1)antisense lncRNA (lncFLI1, ASHGA5P026051), GATA binding protein 2(GATA2) antisense lncRNA (lncGATA2, ASHGA5P019223, RP11-475N22.4),endothelial converting enzyme 1 (ECE1) intron senseoverlapping lncRNA(lncECE1, ASHGA5P032664, AX747766), endothelial cell-selective adhesionmolecule (ESAM) bidirectional lncRNA (lncESAM, ASHGA5P021448,RP11-677M14.3), roundabout homolog 4 (ROBO4) nature antisense RNA(lncROBO4, ASHGA5P026882, RP11-664121.5), and epidermal growthfactor-like domain 7 (EGFL7) intronic antisense lncRNA (lncEGFL7OS,ASHGA5P045551, RP11-251M1.1) were chosen because of their EC enrichmentand potential relevance to EC function. As shown in FIG. 2A, theinventors found that the expression of lncECE1, lncGATA2, lncESAM,lncROBO4, lncFLI1 and lncEGFL7OS was highly enriched in EC cell linescompared to the non-EC lines. Among different EC lines, lncECE1 andlncESAM were more enriched in HUVECs, while lncFLI1 and lncEGFL7OS weremore enriched in HCECs, supporting heterogeneity of ECs and suggestingdifferential expression of the lncRNAs in different ECs.

The inventors also used a bioinformatics approach to determine thetissue distribution of the EC-enriched lncRNAs. The tissue expressioninformation of the top 50 EC-enriched lncRNAs was obtained from theStanford Source database. FIG. 2B showed the tissue distribution heatmapof the candidate lncRNAs that have information available. The majorityof the lncRNAs are enriched in the lung and placenta, which are highlyvascularized tissues. Taken together, these partly validated the EC- andvasculature-enrichment of the candidate lncRNAs from the inventors'microarray.

Expression Pattern of lncEGFL7OS in Human Tissues.

Given the involvement of EGFL7/miR-126 locus in regulate angiogenesis,the inventors focused on lncEGFL7OS, which partially overlaps withEGFL7/miR-126 gene but is transcribed in opposite direction (FIG. 3B).The existence of lncEGFL7OS was confirmed by RT-PCR cloning using humanplacental RACE-ready cDNAs and subsequent sequencing, and the size oflncEGFL7OS is consistent with deposited gene AF161442. Interestingly,conserved homologous sequence of lncEGFL7OS only exists in humans andseveral other primates, including chimpanzee, orangutan and rhesusmonkey, but not in other lower vertebrate species including mice,suggesting lncEGFL7OS is an evolutionarily new gene in mammals. Theinventors performed real-time RT-PCR to examine the tissue expressionpattern of lncEGFL7OS. The inventors found lncEGFL7OS to be highlyenriched in the human lung, placenta and heart, which are highlyvascularized tissues. This is consistent with its EC-enriched expressionpattern from the inventors' array. Since lncEGFL7OS overlaps withEGFL7/miR-126, the expression of EGLF7 and miR-126 was also examined inparallel to lncEGFL7. Human EGFL7 has four isoforms, which the inventorsnamed as EGFL7A to EGFL7D (FIG. 3B). By RT-PCR, EGFL7B and EGFL7C arethe major isoforms expressed in human tissues (FIG. 9). Between thesetwo, EGFL7B is more enriched in the lung, bone marrow and brain, whileEGFL7C is more enriched in the lung compared to other organs, suggestinga differential expression pattern of EGFL7 isoforms in humans. Byreal-time RT-PCR, miR-126 is highly enriched in the bone marrow, lungand heart. Taken together, these results suggest there are both commonand distinct regulatory mechanisms controlling the expression lncEGFL7OSand EGFL7/miR-126 in different human tissues.

Regulation of Angiogenesis by lncEGFL7OS In Vitro and In Vivo.

To define the potential role for lncEGFL7OS in angiogenesis, theinventors have designed three independent siRNAs to knockdown lncEGFL7OSin HUVEC cells, and found two of them successfully silenced lncEGFL7OSexpression, while the third one failed to influence lncEGFL7OSexpression level (FIG. 10). In vitro Matrigel assay was performed toexamine the requirement of lncEGFL7OS in EC tube formation. lncEGFL7OSor control siRNA transfected HUVECs were cultured on Matrigel for 6-8hours, and the primary vascular tubular network was imaged andquantified. Compared to the control, silencing of lncEGFL7OS by twoindependent siRNAs disrupted tube formation as shown by stagnant cells,less organized network, as well as the significant reduced number ofbranch points by quantification (FIGS. 4A-B). As another control, thethird lncEGFL7OS siRNA that failed to silence lncEGFL7OS did not affecttube formation in vitro. These results suggest that lncEGFL7OS isrequired for proper tube formation in ECs.

To further determine the role for lncEGFL7OS in vasculogenesis andangiogenesis, an EC fibroblast co-culture assay was performed. When ECsare cultured on the top of a confluent fibroblast cell layer, ECs willproliferate to form “islands” of ECs, and then sprout to formthree-dimensional vascular tubules resembling capillaries which can bevisualized by immunostaining with an antibody to EC-enriched vonWillebrand factor (vWF) (FIG. 4C). Compared to the control siRNA,si-lincEGFL7OS#1 or si-lncEGFL7OS#2 significantly repressed theformation of vascular tubules at 7 days after co-culture as shown by vWFstaining and the subsequent quantification of the vessel density (FIGS.4C-D). This repressive effect persisted at day 9, although the effect ofsi-lncEGFL7OS#1 is not statistically significant (p=0.059). Takentogether, the inventors conclude that lncEGFL7OS is required for properangiogenesis in vitro.

To examine the requirement of lncEGFL7OS in vasculogenesis/angiogenesisin vivo, si-lncEGFL7OS or control transfected HUVEC cells were mixedwith Matrigel and injected subcutaneously on the back midline of thenude mice, and the primary vascular network was stained with antibodyagainst human CD31 at 14 days after Matrigel implantation. Compared toelongated and connected ECs in the controls, the lncEGFL7OS-silenced ECsappeared rounded and isolated (FIGS. 4E-F). These results indicate thatlncEGFL7OS is required for proper angiogenesis in vivo.

Regulation of EC Proliferation and Migration by lncEGFL7OS In Vitro.

To dissect the cellular mechanism whereby lncEGFL7OS regulatesangiogenesis, a BrDU incorporation assay was carried out to analyze ECproliferation upon lncEGFL7OS silencing. Under starvation condition,si-lincEGFL7OS#2 significantly decreased EC proliferation as shown byBrDU incorporation compared to the random control, while the effect fromsi-lncEGFL7OS#1 was not statistically significant (FIG. 5A). However,the EC proliferation induced by VEGF treatment was significantlyrepressed by either si-lncEGFL7OS#1 or si-lncEGFL7OS#2. To furthercharacterize the reduced EC proliferation after lncEGFL7OS knockdown,the cell cycle profile was quantified after flow cytometry under normalculture conditions. A significant increase in the percentage of cells inthe G0/G1 phase was observed upon lncEGFL7OS knockdown (FIG. 5B).Accordingly, cells in the S and G2/M phase are significantly decreased.This indicates a G1 arrest in the treated cells. The inventors alsodetermined whether EC migration is affected by lncEGFL7OS knockdown.Using a scratch wound assay, the inventors found that compared to thecontrol, lncEGFL7OS silencing significantly repressed EC migration inresponse to VEGF treatment after wound scratch (FIGS. 5C-D). To assesswhether lncEGFL7OS silencing results in EC death, TUNEL assay wasperformed. In the control condition, ˜0.4% of EC cells undergo celldeath, silencing of lncEGFL7OS by siRNA#1 and #2 significantly increasedEC death to ˜0.55% and ˜0.64%, respectively (FIG. 5E). Therefore, theincrease of EC death by si-lncEGFL7OS is statistically significant, butprobably not biologically important with regard to the angiogenicphenotypes observed. These results indicate that lncEGFL7OS is requiredfor proper EC proliferation, migration in vitro.

Regulation of EGFL7/miR-126 Expression by lncEGFL7OS.

LncRNAs could exert regulatory function in cis on the neighboring genes.Since lncEGFL7OS is located in the antisense direction of the regulatoryregion of EGFL7/miR-126, the inventors surmised that lncEGFL7OSregulates angiogenesis by controlling EGFL7/miR-126 expression. Theexpression of EGFL7A, EGFL7B and miR-126 was examined by real-timeRT-PCR upon lncEGFL7OS knockdown. As shown in FIG. 6A, EGFL7B and Cexpression was dramatically decreased upon lncEGFL7OS knockdown. Thedownregulation of EGFL7 at protein level by lncEGFL7OS knockdown wasconfirmed by Western blot analysis (FIG. 6B). Similarly, the expressionof both miR-126 and miR-126*, a microRNA located in the intron of EGFL7gene, is also downregulated by lncEGFL7OS knockdown (FIG. 6C). Asanother control, miR-24 expression was not affected by lncEGFL7OS,suggesting the effect of lncEGFL7OS is specific. miR-126 has been shownto enhance MAP kinase signaling and PI3K-AKT signaling by targetingSpred-1 and PI3KR2, respectively. Accordingly, phosphorylation of ERK1/2and AKT induced by VEGF was significantly reduced in ECs transfectedwith si-lncEGFL7OS#1 or si-lncEGFL7OS#2 compared to the controls (FIG.6D). These results indicate that lncEGFL7OS functions as anenhancer-like noncoding RNA that enhance the expression ofEGLF7/miR-126, and therefore promoting VEGF signaling through MAPK andPI3K/AKT pathways.

Requirement for lncEGFL7OS in Human Choroid Sprouting Angiogenesis ExVivo.

To further explore the function of lncEGFL7OS in angiogenesis in humantissues, the inventors have developed a unique human choroid sproutingassay. Briefly, human choroids were dissected from the donor eyes fromthe eye bank, and were cut into approximately 4 mm2 pieces andtransfected with control or lncEGFL7OS siRNAs overnight. The choroidswere then seeded in the matrigel and cultured in EGM-2 medium for up to10 days. Silencing of lncEGFL7OS by siRNAs (a mix of siRNA #1 and 2 athalf concentration used for other assays) in the system was confirmed byreal-time RT-PCR (FIG. 7A). In the control choroid, significantsprouting was observed at day 10 with an average distance of ˜1200 μm(FIGS. 7B-C). The EC identity of the sprouting cells was confirmed byICAM-2 and Isolectin B4 co-staining (FIG. 7D). Compared to the control,lncEGFL7OS siRNAs drastically repressed human choroid sprouting,establishing a critical role for lncEGFL7OS in angiogenesis in humantissues.

Example 3—Discussion

In this study, the inventors have identified ˜500 EC-enriched lncRNAs bycomparing the lncRNA/mRNA profile from EC and non-EC lines. The EC- orvasculature-5 enrichment of a list of candidate lncRNAs was confirmed byreal-time RT-PCR and bioinformatics approaches. The inventors furtherreported a primate-specific EC-enriched lncEGFL7OS that is located inthe anti-sense strand of EGFL7/miR-126 gene. Silencing of lncEGFL7OSrepresses EC proliferation and migration, therefore impairing vasculartube formation in vitro and in vivo, as well as human choroid sproutingangiogenesis ex vivo. Mechanistically, lncEGFL7OS is required for properactivation of angiogenic signaling at least partly by regulatingEGFL7/miR-126 expression through repressing the methylation ofEGFL7/miR-126 promoter.

Identification of EC-Enriched lncRNAs.

This is the first study to investigate the lncRNA expression profile inECs in comparison to that in non-ECs. The inventors found 498 lncRNAsare enriched in three different primary EC lines compared to non-EClines using a cutoff of 2. By hierarchical cluster analysis,lncRNA-based clustering appeared to be a stronger classifier for EClines than mRNA clustering. This is consistent with the generalperception that lncRNAs exhibit better tissue specificity than mRNAs.The inventors also found significant variability in lncRNA expressionamong EC lines, consistent the observed heterogeneity among ECs. Giventhe central importance of ECs in vascular biology, this dataset mayprovide a foundation to study the regulation and function for lncRNAs invarious vascular development and disease models. Of note, the inventorsalso found many lncRNAs are highly expressed in ECs, but those lncRNAare not necessary EC-specific. Those lncRNAs may also important functionin cell types including ECs.

Looking deep into the gene list, 91 lncRNAs of the 498 EC-enriched geneshave protein coding genes within 10 kB, and about a third of them showedparallel or inverse expression pattern to the associated genes.Functional enrichment analysis indicates that EC-enriched lncRNAs areassociated with genes involved in vascular development. Those lncRNAsmay be good candidates for immediate functional studies.

lncEGFL7OS is a Primate-Specific EC-Enriched lncRNA that is Required forProper Human Angiogenesis.

The inventors discovered lncEGFL7OS that is located in the anti-sensedirection of the EGFL7/miR-126 gene. The expression of this lncRNA isenriched in ECs and highly vascularized tissues, which is consistentwith the expression of its host genes EGFL7 and miR-126. However, thecommon and distinct regulatory mechanisms that direct the expression oflncEGFL7OS, EGFL7 isoforms and miR-126 are still yet to be defined. Theinventors found that lncEGFL7OS is required for proper vascular tubeformation by using Matrigel assay in vitro and in vivo. Using a humanchoroid sprouting angiogenesis model the inventors developed, theinventors further showed that lncEGFL7OS is required for human sproutingangiogenesis. This study indicates that three different transcripts fromthe EGFL7/miR-126 locus, including lncEGFL7OS, EGFL7 and miR-126, haveimportant functions in angiogenesis. EGFL7 and miR-126 have beenpreviously shown to regulate angiogenesis.

EGFL7 is essential for vascular tube formation during vasculogenesis inzebrafish. Deletion of EGFL7 in mice resulted in several vasculardevelopmental defects and partial embryonic lethality, although thisphenotype was attributable by miR-126 loss-of-function by a following upstudy. The importance of miR-126 in angiogenesis was demonstrated byloss-of-function studies in both mouse and zebrafish. Targeted deletionof miR-126 in mice or miR-126 knockdown in zebrafish resulted in loss ofvascular integrity and defective angiogenesis. It is intriguing that, incontrast to EGFL7 and miR-126, lncEGFL7OS represents a primate-specificmechanism in regulating angiogenesis, since lncEGFL7OS only exists inhuman and potentially several other primates. New angiogenesisregulation mechanism through lncEGF7OS has evolved during evolutionunderscores the importance and delicacy of EFGL7/miR-126 locus inangiogenesis. This study also underscores the importance of using human(or primate) system to study the mechanism of angiogenesis.

Mechanism of lncEGFL7OS Action.

The inventors showed that the action of lncEGFL7OS reflects at leastpartially the regulation of expression of EGFL7 and miR-126. miR-126 hasbeen shown to promote MAP kinase and PI3K signaling in response to VEGFand FGF by targeting negative regulators of these signaling pathways,including the Srpouty-related EVH domain-containing protein Spred-1 andPI3K regulatory subunit 2 (PIK3R2). Consistent with the downregulationof miR-126 by lncEGFL7OS silencing, the inventors found that thephosphorylation of ERK1/2 and AKT in response to VEGF is repressed bylncEGFL7OS silencing. Mechanistically, lncEGFL7OS acts an enhancer-likelncRNA that promotes EGFL7/miR-126 expression through repressingmethylation in EGFL7/miR-126 promoter. The full mechanism of lncEGF7OSawaits further studies.

Therapeutic Implications.

Identifying angiogenic mechanisms that are conserved to human orhuman-specific is critical for developing therapeutics for humanvascular disorders. These studies have demonstrated that lncEGFL7OS is aprimate-specific lncRNA critical for human angiogenesis. This isdirectly translatable for human diseases involving abnormalangiogenesis: particularly for AMD, the leading cause of blindness inthe elderly, and choroidal neovascularization, a hallmark for wet AMD.Although anti-VEGF agents can markedly improve the clinical outcome ofwet AMD, they have been unable to induce complete angiogenesisregression, and only 30-40% of individuals experienced visionimprovement after treatment. The inventors developed a human choroidsprouting angiogenesis model and showed that lncEGFL7OS represses humanchoroid sprouting angiogenesis.

Example 4—Materials and Methods

Animals and In Vivo Angiogenesis Assay.

Animal studies were conducted in accordance with the ARVO statement forthe Use of Animals in Ophthalmic and Vision Research and were approvedby the Institutional Animal Care and Use Committees at the TulaneUniversity. BALB/cAnN-nu (Nude) mice (6 to 8 weeks of age) from Jacksonlab were used for in vivo angiogenesis assay. In vivo Matrigel analysiswas performed. HUVEC cells transfected with control si-RNA, or mix ofsi-LncEGFL7OS#1 and si-LncEGFL7OS#2 (50 nM each) for 2 days. Cells werethen trypsinized and about 5×10⁵ cells were mixed with 50 μl EMB-2 mediaand 350 μl ice-cold Matrigel (BD Biosciences). The mixture was thenapplied under the back skin of 8 week-old BALB/cAnN-nu (Nude) femalemice (Jackson lab). After 14 days, The Matrigel plugs were extracted andsnap-frozen in OCT and processed for immunostaining with human EC markerPECAM-1 and tube length quantification.

Cell Culture.

HUVEC (ATCC) cells were grown in EC growth medium EGM-2 (Lonza). HCECand HREC cells were kindly provided by Dr. Ashwath Jayagapol fromVanderbilt University and grown in EGM2 media (Lonza). ARPE-19 (ATCC)cells were growth in DMEM/F12 (HyClone) media with 10% FBS. HDF (ATCC)cells were grown in DMEM (HyClone) with 10% FBS. For VEGF treatment,HUVECs were starved with EC basal medium-2 with 0.1% FBS for 24 h andthen treated with VEGF (20 ng/mL) for the indicated periods of time.SiRNA transfection in cell culture was performed as described (Zhou etal., 2013). SiRNAs for LncEGFL7OS were purchased from sigma. Sequencesfor siRNAs are as follows: si-lncEGFL7OS#1:5′-GCGUUUCCCUAGCAAUGUUdTdT-3′ (SEQ ID NO: 2) and5′-AACAUUGCUAGGGAAACGCdTdT-3′ (SEQ ID NO: 3); silncEGFL7OS#2:5′-CAGCUUUGCCCUAUCCCAUdTdT-3′ (SEQ ID NO: 4) and5′-AUGGGAUAGGGCAAAGCUGdTdT-3′ (SEQ ID NO: 5).

LncRNA Microarray.

RNAs from five cell lines were purified by MirVana™ total RNA IsolationKit (Ambion, Invitrogen). These RNAs were subjected to microarray-basedglobal transcriptome analysis (Arraystar Human LncRNA array (version2.0), Arraystar Inc, Rockville, Md.). This LncRNA microarray is designedto detect about 30,586 LncRNAs and 26,109 coding transcripts. TheLncRNAs were constructed using the most highly-respected publictranscriptome databases (Refseq, UCSC known genes, Gencode, etc.), aswell as landmark publications. The lncRNA probes include 19590intergenic lncRNAs (lincRNAs), 4409 intronic lncRNAs, 1299 bidirectionallncRNAs, 1597 sense overlapping lncRNAs and 3691 antisense lncRNAs. Dataanalyses, including hierarchy clustering analysis and functionalenrichment analysis, were performed using Genescript software. Tissuedistribution data of the top-50 candidates was downloaded from StanfordSource database.

Cell Proliferation, Cell Cycle Analysis, TUNEL Assay, Scratch-Wound, InVitro Matrigel assays.

EC cell proliferation, TUNEL assay and scratch-wound assays wereperformed using HUVEC cells as described. For cell proliferation assay,about 2×10³ transfected HUVECs were seeded in 96-well plates. Afterstarvation with 0.1% serum for overnight, the cells were stimulated with20 ng/mL VEGF-A for 20 hours and then subjected to BrDU labeling for 4hours. DNA synthesis as determined by BrDU incorporation was quantifiedusing a commercial ELISA kit from Roche according to the manufacturer'sinstructions. Cell cycle analysis was performed using Guava® Cell CycleReagents (Guava Technologies) on a Guava instrument and analyzed usingCytosoft™ software according to the manufactural manual. For scratchwound assay, scratch-wound was made using a 200-μL pipette tip in lncRNAor control siRNA-transfected HUVEC monolayer before VEGF (20 ng/ml)stimulation. 1 μM 5-fluouracil (Sigma) was then added to the cells rightafter scratch wound to block cell proliferation. Post-scratch ECmigration was scored at 14 h after wound scratch. For in vitroangiogenesis assay, at 3 days after lncRNA or control siRNA transfectionwith Liptofectamine RNAiMAX™ reagent (Invitrogen), cells were harvestedfor RNA or in vitro Matrigel assay and branch point analysis asdescribed before.

In Vitro EC-Fibroblast Co-Culture Angiogenesis Assay.

In vitro EC-Fibroblast co-culture was performed as described. Briefly,HDF were seeded into each well of a 12-well plate and maintained in DMEMwith medium changes every 2-3 days until they developed confluentmonolayers. HUVECs were maintained as described above and transfectedwith siRNA one day prior to seeding on HDF monolayers. Approximately3×10⁴ HUVECs were seeded onto each monolayer and the HDF/HUVECco-culture was maintained for 7-14 days in EGM-2 medium with mediumchanges every 2-3 days to allow endothelial cell polarization,migration, networking, and the formation of an in vitro primitivevascular plexus.

After 7-14 days the wells were fixed with 100% Methanol at −20° C. for20 minutes and then stained with anti-VWF antibody (Dako). Afterhybridizing a secondary antibody, the endothelial tissue was visualizedand imaged under a Nikon microscope. Multiple images were automaticallystitched with Nikon software to provide a large image (several mm²) andthe resulting image was analyzed on ImageJ software to determine thedegree of vascularization. Three wells were used for each condition andresults are representative of the mean of each three-well group. Theexperiments were repeated for at least three repeats with similarresults.

Ex Vivo Human Choroid Sprouting Assay.

Ex vivo human choroid sprouting assay was adapted from a mouse protocol.Donated human eye balls were obtained from Southern eye bank (NewOrleans, La.). Eyes were cleaned and kept in sterile ice-cold PBS withPenicillin/Streptomycin before dissection. Using fine forceps, thecornea and the lens from the anterior of the eye were removed. Theperipheral choroid-scleral complex was separated from the retina and theRPE layer was peeled away using fine forceps. The choroid-scleralcomplex was then cut into approximately 1 mm×1 mm pieces using sterilescalpel blade under laminar airflow. The choroid was then washed withsterile ice-cold PBS and transferred into endothelial base medium (EBM2)with 0.1% FBS (300 μl/well in 24-well plates). The choroid wastransfected with control si-RNA, or mix of si-LncEGFL7OS#1 andsi-LncEGFL7OS#2 (50 nM each) for overnight. Choroid fragments were thenwashed by EGM2 media then placed in growth factor-reduced Matrigel™ (BDBiosciences) in 24-well plate. Briefly, 30 μl of matrigel was used tocoat the bottom of 24 well plates without touching the edge of the well.After seeding the choroid, the plate was incubated in a 37° C. cellculture incubator to make the Matrigel solidify. 500 μl EC growth medium(EGM-2 with 3% of mouse serum) were added slowly to the plate withoutdisturbing the Matrigel, and the plate was incubated at 37° C. cellculture incubator with 5% CO₂. Cell culture medium was changed every 48hours. The EC sprouts normally start to appear on the day 5 and growrapidly between day 8 and 10. Phase contrast photos of individualexplants were taken using a Nikon microscope. The sprouting distance wasquantified with computer software ImageJ (National Institute of Health).Sprouting ECs were stained with ICAM-2 or isolectin B4.

RNA, Western Blot Analysis and Immunofluorescence.

Human total RNA master panel II was purchased from clontech (Takara).Total RNA was isolated from human choroid tissues or cell lines usingTRIzol reagent (Invitrogen). Cytoplasmic and nuclear RNA was purifiedusing a Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek Corp.,Thorold, ON, Canada) according to manufacturer's supplied protocol. Inbrief, cells growing in monolayer were rinsed with 1×PBS and lyseddirectly on the plate with ice-cold Lysis Buffer. Next cell lysate wastransfer to the RNase-free microcentrifuge tube and spun for 3 minutesat 14,000×g. Supernatant containing cytoplasmic RNA was mixed withmanufacturer's supplied buffer (Buffer SK) and 100% ethanol, and appliedonto a spin column. The pellet containing the nuclear RNA was mixed withBuffer SK and 100% ethanol, and applied onto a spin column. Both columnswere washed with supplied Wash Solution, and RNA was eluted withsupplied elution buffer (Elution Buffer E). For maximum recovery tworounds of elution were performed. Quantitative (q) RT-PCR or regularRT-PCR was performed using iScript™ cDNA Synthesis system (BioRad),miRNA qRT-PCR was performed using gScript™ cDNA Synthesis and microRNAQuantification System (Quanta Biosciences). Primers for real-time PCRsinclude human!-actin, 5′-GAGCAAGAGATGGCCACGG-3′ (SEQ ID NO: 6) and5′-ACTCCATGCCCAGGAAGGAA-3′ (SEQ ID NO: 7); lnc-FLI1-AS1 (also namedSENCR), up: 5′-CCTGAGGCCATCTTACCACC-3′ (SEQ ID NO: 8), down:5′-AATCCGCTTCGATGAGTGGG-3′ (SEQ ID NO: 9); SENCR (for regular PCR), up:5′-GCGCATTGTTAGGAGAAGGG-3′ (SEQ ID NO: 26), down:5′-CCTGCTGACTGTCCTAGAGG-3′ (SEQ ID NO: 27); lnc-GATA2-AS, up:5′-CGGGCAGCTTACGATTCTTC-3′ (SEQ ID NO: 10), down:5′-CGGTGTCTTTCAGAGGGTCT-3′ (SEQ ID NO: 11); lnc-ECE1, up:5′-CCATGTCGCCTCAGCCTAAA-3′ (SEQ ID NO: 12), down:5′-GGGCAGTCTCAGGGTAACAC-3′ (SEQ ID NO: 13); lnc-ESAM, up:5′-CTCGGAAAACGGAGGGTTGA-3′ (SEQ ID NO: 14), down:5′-CGCTGCCCTTAATTCCTTGC-3′ (SEQ ID NO: 15); lnc-ROBO4-AS, up:5′-ACCAGCAGACCCTGAAACTC-3′ (SEQ ID NO: 16), down:5′-GGCAGGGATCAGGCATTCAT-3′ (SEQ ID NO: 17); lnc-EGFL7-AS, up:5′-AGTGCCAGCTTTGCCCTATC-3′ (SEQ ID NO: 18), down:5′-GAGAACACAGGACGTCCACA-3′ (SEQ ID NO: 19); EGFL7-A, up:5-CTTCAGAGGCCAAAAGCACC-3′ (SEQ ID NO: 20), down:5′-GAATCAGTCATCCCCCGGAC-3′ (SEQ ID NO: 21); EGFL7-B, up:5′-AAGGGAGGCTCCTGTGGA-3′ (SEQ ID NO: 22), down: 5′-CCTGGGGGCTGCTGATG-3′(SEQ ID NO: 23); EGFL7-C, up: 5′-CGGATCCGGCGGCCA-3′ (SEQ ID NO: 24),down: 5′-CGAACGACTCGGAGACAGG-3′ (SEQ ID NO: 25); Neat1, up:5′-AGATACAGTGTGGGTGGTGG-3′ (SEQ ID NO: 28), down:5′-AGTCTTCCCCACCTTGTAGC-3′ (SEQ ID NO: 29). Human primiR-126, up:5′-TGGCGTCTTCCAGAATGC-3′ (SEQ ID NO: 30), down: 5′-TCAGCCAAGGCAGAAGT-3′(SEQ ID NO: 31). For Western blot analysis, protein lysates wereresolved by SDS-PAGE and blotted using standard procedures. Antibodiesused were as follows: ERK1/2 (Cell signaling), Phospho-ERK1/2 (Cellsignaling), AKT (Cell signaling), Phospho-AKT (Cell signaling), EGFL-7(Abcam) and β-Tublin (Abcam). For immunofluorescence experiments,samples were fixed with 4% paraformaldehyde or methanol for 30 min.After treatment with 1% Triton X-100 in PBS, samples were incubated inPBS containing 4% goat serum for 30 min. The samples were then incubatedwith primary antibodies overnight at 4° C., followed by incubation withappropriate secondary antibodies. Antibody used for immunofluorescenceinclude: vWF (DAKO), ICAM2 (BD Pharmingen), PECAM-1 (DAKO).

High Resolution RNA FISH Experiments.

QuantiGene® (QG) ViewRNA ISH Cell Assay reagents (Affymetrix) were usedfor RNA Fluorescence In Situ Hybridization (FISH). Custom probeoligonucleotide pair pools specific for lncEGFL7, PP1B and NEAT1 weredesigned and synthesized by Affymetrix. RNA-FISH was performed followingthe manufacturer's protocol with minor modifications: HUVEC cell weregrown on acid-washed #1.5 glass cover slips to 70%-80% confluence,washed with PBS, and fixed for 30 min in 4% paraformaldehyde. Afterpermeabilization using QG Detergent Solution, cells were treated with0.5% Triton X-100/PBS for 5 min at room temperature. Partial proteasedigestion was carried out with a 1:6,000 dilution of QG Protease K for10 min at room temperature. Cells were incubated with primary probe pairsets at 40° C. for 3 hr. Pre-amplifiers were incubated for 1 hr at 40°C. Between probe set incubations, cells were washed 4 times each in QGWash Buffer for a total of 10 min. After counter-staining with DAPI,coverslips were mounted in mounting medium and sealed with nail polish.Pictures were taken under a Nikon A1 confocal microscope.

Chromatin Immunoprecipitation (ChIP) Assay.

ChIP-IT Express kit (Active Motif) were used for ChIP assay. HUVEC cellsgrow up to 70-80% confluency in 15 cm plate for ChIP assay. The ChIPassay was performed according to manufacturer's protocol. ChIP gradeantibodys used in ChIP assay: Max (Santa Cruz, sc-197), Myc(Sigma-Aldrih, c3956), Anti-RNA Polymerase II (Abcam, ab5408),Tri-Methel-Histon H3 (Lys4) (Cell Signaling, #9751), ETS1 (Santa Cruz,sc-111), Normal Rabbit IgG (Cell Signaling, #2729). ChIP samples wereanalyzed by using normal PCR with following parameters: (1) initialdenaturation at 94° C. for 10 min, (2) denaturation at 94° C. for 20 s,(3) anneal at 58° C. for 30 s, (3) extension at 72° C. for 1 min. Stepsfrom 2 to 4 were repeated 35 times. Primers to amplify conservedtranscription factors binding region in the lncEGFL7OSregulator/promoter region and control region were as follows: primers1(regulatory region), 5′-CCTCCTGTTTGTCCGACGAC-3′ (SEQ ID NO: 32) and5′-GGAAGGGCGGCTTTTTATGC-3′ (SEQ ID NO: 33); primers2 (control region),5′-AGATCCCAGGGCTGTTTAGC-3′ (SEQ ID NO: 34) and5′-AACACTCCTCCCAGCGAATC-3′ (SEQ ID NO: 35).

Luciferase assay. Luciferase assays were performed. The putativebidirectional promoter for lncEGFL7OS/EGFL7 was PCR amplified from humanDNA and cloned into promoterless PGL3 Basic luciferase vector (Promega).Primers include: pincEGFL7OSup (XhoI):5′-atcgCTCAGATAGACTCTGATGGCCCAGG-3′ (SEQ ID NO: 36) and pincEGFL7OSdn(XhoI): 5′-atcgCTCAGACCAGCTTGGTGCAGGGAG-3′ (SEQ ID NO: 37). 293T cellsin 24-well plates were transfected with 50 ng of reporter plasmids inthe presence or absence of increasing amount of Ets1 or Ets1

DNA-binding mutant expression plasmid.

Statistics.

Each experiment was repeated at least three times. Student's t-testswere used to determine statistically significant differences betweengroups. P-values of less than 0.05 were considered to be statisticallysignificant.

Example 5—Results

Confirmation of the EC-Enriched lncRNAs.

Quantitative (q) RT-PCR was used to confirm a selected list ofEC-enriched lncRNAs from the microarray. Friend leukemia integration 1(FLI1) antisense lncRNA (FLI1AS, also named as SENCR (18),ASHGA5P026051), GATA binding protein 2 (GATA2) antisense lncRNA(lncGATA2, ASHGA5P019223, RP11-475N22.4), endothelial converting enzyme1 (ECE1) intron sense-overlapping lncRNA (lncECE1, ASHGA5P032664,AX747766), endothelial cell-selective adhesion molecule (ESAM)bidirectional lncRNA (lncESAM, ASHGA5P021448, RP11-677M14.3), roundabouthomolog 4 (ROBO4) nature antisense RNA (lncROBO4, ASHGA5P026882,RP11-664121.5), and epidermal growth factor-like domain 7 (EGFL7)opposite strand lncRNA (lncEGFL7OS, ASHGA5P045551, RP11-251M1.1) werechosen because of their EC enrichment and potential relevance to ECfunction. As shown in FIG. 12A, the expression of lncECE1, lncGATA2,lncESAM, lncROBO4, lncFLI1 and lncEGFL7OS was found to be highlyenriched in EC cell lines compared to the non-EC lines. Among differentEC lines, lncECE1 and lncESAM were more enriched in HUVECs, while FLI1ASand lncEGFL7OS were more enriched in HCECs, supporting heterogeneity ofECs and suggesting differential expression of the lncRNAs in differentECs.

The inventors also used a bioinformatics approach to determine thetissue distribution of the EC-enriched lncRNAs. The tissue expressioninformation of the top 50 EC-enriched lncRNAs was obtained from theStanford Source database. FIG. 12B showed the tissue distributionheatmap of the candidate lncRNAs that have information available. Themajority of the lncRNAs are enriched in the lung and placenta, which arehighly vascularized tissues. Taken together, these partly validated theEC- and vasculature-enrichment of the candidate lncRNAs from theinventors' microarray.

Expression Pattern of lncEGFL7OS in Human Tissues.

Given the involvement of EGFL7/miR-126 locus in regulating angiogenesis,the inventors focused on lncEGFL7OS, which partially overlaps withEGFL7/miR-126 gene but is transcribed in opposite direction (FIG. 12B).The existence of lncEGFL7OS was confirmed by RT-PCR cloning using humanplacental RACE-ready cDNAs and subsequent sequencing, and the size oflncEGFL7OS is consistent with deposited gene AF161442. Interestingly,conserved homologous sequence of lncEGFL7OS only exists in humans andprimates Rhesus monkey, but not in other lower vertebrate speciesincluding mice, suggesting lncEGFL7OS-AS is an evolutionarily new genein mammals. The inventors performed qRT-PCR to examine the tissueexpression pattern of lncEGFL7OS.

LncEGFL7OS was found to be highly enriched in the human lung, placentaand heart, which are highly vascularized tissues (FIG. 12C). This isconsistent with its EC-enriched expression pattern from the inventors'array. Since lncEGFL7OS overlaps with EGFL7/5 miR-126, the expression ofEGLF7 and miR-126 was also examined in parallel to lncEGFL7OS. HumanEGFL7 has four isoforms, named as EGFL7A-D, but only EGFL7B and EGFL7Care detectable by RT-PCR in human tissues (FIG. 12D). Between these two,EGFL7B is more enriched in the lung, bone marrow and brain, while EGFL7Cis more enriched in the lung compared to other organs, suggesting adifferential expression pattern of EGFL7 isoforms in humans. By qRT-PCR,miR-126 is highly enriched in the bone marrow, lung and heart (FIG.12E). Taken together, these results suggest there are both common anddistinct regulatory mechanisms controlling the expression lncEGFL7OS andEGFL7/miR-126 in different human tissues.

The inventors also examined the subcellular localization of lncEGFL7OSusing both semi-quantitative RTPCR and high-resolution RNA fluorescencein situ hybridization (FISH). By RT-PCR, lncEGFL7OS was shown to beexpressed in both the cytoplasm and nucleus, but predominantly in thenucleus of HUVECs (FIG. 12F). SENCR was used a marker forcytoplasmic-enriched lncRNA, while NEAT-1 was used as a marker fornuclear enriched-lncRNA. These results were confirmed by high-resolutionRNA FISH experiment. RNA FISH with single-molecule sensitivity wasperformed using oligonucleotide (oligo) probe pools specific forlncEGFL7OS. The inventors observed variably low numbers of lncEGFL7OSmolecules in both the nucleus and cytoplasm of HUVECs, which contrastswith the high level expression of nucleus-enriched NEAT1 lncRNA andcytoplasmic-enriched PP1B mRNA (FIG. 12G). Taken together, these dataindicate that lncEGFL7OS is expressed at low copy numbers in both thenucleus and cytoplasm of HUVEC cells.

Regulation of lncEGFL7OS Expression by ETS Factors Through aBidirectional Promoter in HUVECs.

To dissect the lncEGFL7OS regulation mechanism in relation to its hostgene EGFL7/miR-126, the inventors aimed to identify the potentialregulatory elements for lncEGFL7OS. The inventors have analyzed the celltype-specific active element of the locus from online database UCSCgenome browser (FIG. 13A). A critical regulatory element is located onEGFL7B promoter between lncEGFL7OS and miR-126. Bioinformatics data fromENCODE indicate that LncEGFL7OS DNA contains a region positive forepigenetic marks including high histone H3 trimethylated lysine 4methylation (H3K4Me1) and H3K27Ac (mark active and poised enhancers),low H3K4Me3 (marks promoter of protein coding genes), and binding sitesfor transcription factors MAX, MYC and RNA Polymerase (PolR) II(although PolR II binding to region1 is much weaker compared to region2) (FIG. 13A). Several binding sites for ETS transcription factors werefound in region. Homologous region drives the EC-enriched LacZ reportergene expression in vivo.

Consistently, chromatin immunoprecipitation (ChIP) PCR assay usingantibodies against MAX/MYC, RNA Pol II and histone H3 trimethylatedlysine 4 (H3K4me3) demonstrated the binding of these factorsspecifically to the region but not a non-relevant nearby region,indicating that this region is transcriptional active (FIG. 13B).Additional potential promoters were not found in the region betweenlncEGFL7OS and EGFL7 transcripts by bioinformatics approach. Instead,CpG islands were found in the region. CpG islands in mammalian promoterregions tend to show bidirectional promoter activity. Bidirectionalpromoters have been proposed to drive head-to-head gene transcriptioninvolving ncRNAs. Based on these, the inventors tested a novelhypothesis that a bidirectional promoter (lncEGFL7OS/EGFL7 promoter)regulated by ETS factors drives the expression of both lncEGFL7OS andEGFL7 expression in human ECs. The putative lncEGFL7OS promoter wascloned into a promoter-less luciferase reporter construct in eithersense or anti-sense direction. By luciferase assay, the promoter ineither directions exhibited similar activity under baseline in 293Tcells (FIG. 13C). Moreover, ETS1 transcription factor, but not theETS1mut that lacks the DNA binding domain, significantly activated thepromoter activity in either direction. ETS factors have been shown toregulate miR-126 expression in ECs. To further test whether ETS factorsare required to regulate lncEGFL7OS expression, ETS1 and ETS2 genes weresilenced in HUVEC cells, and lncEGFL7OS and miR-126 expression wereexamined by qRT-PCR. LncEGFL7OS and pri-miR-126 expression wassignificantly reduced by ETS1/2 silencing, suggesting ETS factorscontrol the expression of both lncEGFL7OS and miR-126 (FIG. 13D).

Regulation of Angiogenesis by lncEGFL7OS In Vitro and In Vivo.

To define the potential role for lncEGFL7OS in angiogenesis, theinventors have designed three independent siRNAs to knockdown lncEGFL7OSin HUVEC cells, and found two of them successfully silenced lncEGFL7OSexpression, while the third one failed to influence lncEGFL7OSexpression level (FIG. 10). In vitro Matrigel assay was performed toexamine the requirement of lncEGFL7OS in EC tube formation. lncEGFL7OSor control siRNA transfected HUVECs were cultured on Matrigel for 6-8hours, and the primary vascular tubular network was imaged andquantified. Compared to the control, silencing of lncEGFL7OS by twoindependent siRNAs disrupted tube formation as shown by stagnant cells,less organized network, as well as the significant reduced number ofbranch points by quantification (FIGS. 14A-B). As another control, thethird lncEGFL7OS siRNA that failed to silence lncEGFL7OS did not affecttube formation in vitro. These results suggest that lncEGFL7OS isrequired for proper tube formation in ECs. To further determine the rolefor lncEGFL7OS in vasculogenesis and angiogenesis, an EC-fibroblastco-culture assay was performed. When ECs are cultured on the top of aconfluent fibroblast cell layer, ECs will proliferate to form “islands”of ECs, and then sprout to form three-dimensional vascular tubulesresembling capillaries which can be visualized by immunostaining with anantibody to EC-enriched von Willebrand factor (vWF) (FIG. 14C). Comparedto the control siRNA, si-lincEGFL7#1 or si-lncEGFL7OS#2 significantlyrepressed the formation of vascular tubules at 7 days after co-cultureas shown by vWF staining and the subsequent quantification of the vesseldensity (FIGS. 14C-D). This repressive effect persisted at day 9,although the effect of si-lncEGFL7OS#1 is not statistically significant(p=0.059). Taken together, the inventors conclude that lncEGFL7OS isrequired for proper angiogenesis in vitro.

To examine the requirement of lncEGFL7OS in vasculogenesis/angiogenesisin vivo, silncEGFL7OS or control transfected HUVEC cells were mixed withMatrigel and injected subcutaneously on the back midline of nude mice,and the primary vascular network was stained with antibody against humanCD31 at 14 days after Matrigel implantation. Compared to elongated andconnected ECs in the controls, the lncEGFL7OS-silenced ECs appearedrounded and isolated (FIGS. 14E-F). These results indicate thatlncEGFL7OS is required for proper angiogenesis in vivo.

Regulation of EC Proliferation and Migration by lncEGFL7OS In Vitro.

To dissect the cellular mechanism whereby lncEGFL7OS regulatesangiogenesis, a BrDU incorporation assay was carried out to analyze ECproliferation upon lncEGFL7OS silencing. Under starvation condition,si-lincEGFL7#2 significantly decreased EC proliferation as shown by BrDUincorporation compared to the random control, while the effect fromsilncEGFL7OS#1 was not statistically significant (FIG. 15A). However,the EC proliferation induced by VEGF treatment was significantlyrepressed by either si-lncEGFL7OS#1 or silncEGFL7OS#2. To furthercharacterize the reduced EC proliferation after lncEGFL7OS knockdown,the cell cycle profile was quantified after flow cytometry under normalculture conditions. A significant increase in the percentage of cells inthe G0/G1 phase was observed upon lncEGFL7OS knockdown (FIGS. 15B-C).Accordingly, cells in the S and G2/M phase are significantly decreased.This indicates a G1 arrest in the si-lncEGFL7OS treated cells. Theinventors also determined whether EC migration is affected by lncEGFL7OSknockdown. Using a scratch wound assay, the inventors found thatcompared to the control, lncEGFL7OS silencing significantly repressed ECmigration in response to VEGF treatment after wound scratch (FIGS.15D-E). To assess whether lncEGFL7OS silencing results in EC death,TUNEL assay was performed. In the control condition, ˜0.4% of EC cellsundergo cell death, silencing of lncEGFL7OS by siRNA#1 and #2significantly increased EC death to ˜0.55% and ˜0.64%, respectively(FIG. 15F). Therefore, the increase of EC death by si-lncEGFL7OS isstatistically significant, but probably not biologically important withregard to the angiogenic phenotypes observed. These results indicatethat lncEGFL7OS is required for proper EC proliferation and migration invitro.

Regulation of EGFL7/miR-126 Expression by lncEGFL7OS.

LncRNAs could exert regulatory function in cis on the neighboring genes.Since lncEGFL7OS is located in the opposite strand neighboringEGFL7/miR-126, the inventors surmised that lncEGFL7OS regulatesangiogenesis by controlling EGFL7/miR-126 expression. The expression ofEGFL7BC and miR-126 was examined by qRT-PCR upon lncEGFL7OS knockdown.As shown in FIG. 16A, EGFL7B and C expression was dramatically decreasedupon lncEGFL7OS knockdown. The downregulation of EGFL7 at protein levelby lncEGFL7OS knockdown was confirmed by Western blot analysis (FIG.16B). Similarly, the expression of both miR-126 and miR-126*, a microRNAlocated in the intron of EGFL7 gene, is also downregulated by lncEGFL7OSknockdown (FIG. 16C). As another control, miR-24 expression was notaffected by lncEGFL7OS, suggesting the effect of lncEGFL7OS is specific.miR-126 has been shown to modulate MAP kinase signaling and PI3K-AKTsignaling by targeting Spred-1 and PI3KR2, respectively. Consistentlywith the downregulation of miR-126, phosphorylation of ERK1/2 and AKTinduced by VEGF was significantly reduced in ECs transfected withsi-lncEGFL7OS#1 or si-lncEGFL7OS#2 compared to the controls (FIG. 16D).These results indicate that lncEGFL7OS functions as an enhancer-likenoncoding RNA that enhance the expression of EGLF7/miR-126, andtherefore promoting VEGF signaling through MAPK and PI3K/AKT pathways.

Requirement for lncEGFL7OS in Human Choroid Sprouting Angiogenesis ExVivo.

To further determine the function of lncEGFL7OS in angiogenesis in humantissues, the inventors have developed a unique human choroid sproutingassay. Briefly, human choroids were dissected from the donor eyes fromthe eye bank, and were cut into approximately 4 mm² pieces andtransfected with control or lncEGFL7OS siRNAs overnight. The choroidswere then seeded in the matrigel and cultured in EGM-2 medium for up to10 days. Silencing of lncEGFL7OS by siRNAs (a mix of siRNA #1 and 2 athalf concentration used for other assays) in the system was confirmed byqRT-PCR (FIG. 17A). In the control choroid, significant sprouting wasobserved at day 10 with an average distance of ˜1200 μm (FIGS. 17B-C).The EC identity of the sprouting cells was confirmed by ICAM-2 staining(FIG. 17D). Compared to the control, lncEGFL7OS siRNAs drasticallyrepressed human choroid sprouting, establishing a critical role forlncEGFL7OS in angiogenesis in human tissues.

Overexpression of lncEGFL7OS Enhances Angiogenesis.

To examine the phenotype of lncEGFL7OS overexpression, the inventorshave generated lncEGFL7OS or control GFP adenoviruses, and used them toinfect HUVEC cells at MOI of 50. Infected ECs were cultured on afibroblast mono layer, and their angiogenic response was examined bystaining with an antibody to PECAM-1 at 9 days after co-culture. Theefficiency of the virus was verified by qRT-PCR. Over 2000-foldlncEGFL7OS expression was achieved in ECs after virus infection at 50multiplicity of infection (MOI) (FIG. 14A). Compared to the GFP control,lncEGFL7OS overexpression enhanced angiogenesis as shown by thesignificantly increased tube length compared to the controls (FIGS.14B-C). These data indicate that overexpression of lncEGFL7OS issufficient to enhance EC angiogenesis.

lncEGFL7OS Enhances EGFL7/miR-126 Transcription by Interacting with MAXTranscription Factor.

To study the mechanism whereby lncEGFL7OS regulates EGFL7/miR-126expression, the inventors hypothesized that lncEGFL7OS regulatesEGFL7/miR-126 promoter activity by interacting with MAX transcriptionfactor. MAX was predicted as one of the top lncEGFL7OS-interactingproteins by lncRNA interaction prediction program catRAPID. It has beenshown to interact with Myc to control cell proliferation and cell death.Online database UCSC genome browser predicts the existence of MAXbinding sites near to EGFL7B but between lncEGFL7OS and EGFL7/miR-126genes (FIG. 15A). ChIP-PCR assays confirmed the specific binding of MAXto this region in ECs (FIG. 15B). Moreover, overexpression of lncEGFL7OSsignificantly increased the binding of MAX to this region. MAX has beenshown to dimerize with MYC, which can stimulate histone acetylation andgene transcription 40. The inventors further examined the enrichment ofacetylated H3K27 (H3K27ac), a marker for active enhancer, in this region(FIG. 15C). H3K27ac was found to be enriched in the region, which wasfurther increased by lncEGFL7OS overexpression. These results suggestthat lncEGFL7OS promotes the binding of MAX protein to the bidirectionalpromoter/enhancer region of EGFL7/miR-126, and enhances theirtranscription. The inventors further tested whether lncEGFL7OS interactswith MAX protein in ECs. RNA immunoprecipitation (RIP) assays showedthat lncEGFL7OS RNA was pulled down in the nuclear lysate by aChip-grade antibody to MAX, and this interaction was increased bylncEGFL7OS overexpression (FIG. 15D). To examine whether MAX is requiredfor regulating lncEGFL7OS/EGFL7/miR-126 expression, two specific siRNAswere used to silence MAX expression (FIG. 15E). MAX silencing resultedin decreased expression of EGFL7, lncEGFL7OS and miR-126 (FIG. 15F-H).Consistently, MAX silencing led to repressed angiogenesis as shown byEC/Fibroblast co-culture assays (FIG. 15I). The inventors furtherdetermine whether MAX silencing overrides the increased expression ofmiR-126 induced by adenovirus expressing lncEGFL7OS. As shown in FIG.15J, the induction of miR-126 expression by lncEGFL7OS overexpressionwas blunted by MAX knockdown. Together, these data indicate thatlncEGFL7OS regulates EGFL7/miR-126 expression by interaction with MAXtranscription factor, which enhances H3K27 acetylation in theEGFL7/miR-126 promoter/enhancer region.

Generation of lncEGFL7OS-KO ES Clones.

LncEGFL7OS has two exons, sized about 100 nt (Exon-1) and 500 nt(Exon-2). At the current stage of lncRNA research, it is impractical toidentify the critical functional regions of lncRNAs usingbioinformatics. Therefore, guide RNAs have been designed to targetExon-1 and/or Exon-2 using the available online tools (crispr.mit.edu/)(FIG. 17A). They will be cloned into lentiviral human sgRNA expressionvector. Lentiviruses from the vectors be used to co-transduce human EScell line WA01 with lenti-dCas9 virus. After puromycin selection, cloneswill be picked up, expanded and screened for bi-allele deletion ofExon-1 and/or Exon-2 by PCR using primers spanning the deletions andsubsequent sequencing. The efficiency of the CRISPR deletion of Exon Iand/or II has been verified in 293T cells (FIG. 17B). Although CRISPRapproach is known to have extremely low off-target, potential off-targetregions predicted by CRISPR online tools will be amplified usinghigh-fidelity DNA polymerase and sequenced. The efficiency oflncEGFL7OS-KO in ES lines will be confirmed by qRT-PCR afterdifferentiation into ECs. Neither EGFL7 nor miR-126 has been shown toimpact EC differentiation. Therefore, the inventors do not expect severedefects in EC differentiation in lncEGFL7OS-KO ES cells. If lncEGFL7-KOaffects EC differentiation, that would indicate an unexpected functionof LncEGFL7OS in that process.

Example 6—Discussion

In this study, the inventors have identified ˜500 EC-enriched lncRNAs bycomparing the lncRNA/mRNA profile from EC and non-EC lines. The EC- orvasculature-enrichment of a list of candidate lncRNAs was confirmed byqRT-PCR and bioinformatics approaches. The inventors further reported aprimate-specific EC-enriched lncEGFL7OS that is located in the oppositestrand neighboring the EGFL7/miR-126 gene. Expression of lncEGFL7OS inECs is regulated by ETS transcription factors through a bidirectionalpromoter. Silencing of lncEGFL7OS represses EC proliferation andmigration, therefore impairing vascular tube formation in vitro and invivo, as well as human choroid sprouting angiogenesis ex vivo.Mechanistically, lncEGFL7OS is required for proper activation ofangiogenic signaling at least partly by regulating EGFL7/miR-126expression.

Identification of EC-Enriched lncRNAs.

To the inventors' knowledge, this is the first study to investigate thelncRNA expression profile in ECs in comparison to that in non-ECs. Theinventors found 498 lncRNAs are enriched in three different primary EClines compared to non-EC lines using a cutoff of 2. By hierarchicalcluster analysis, lncRNAPage based clustering appeared to be a strongerclassifier for EC lines than mRNA clustering. This is consistent withthe general perception that lncRNAs exhibit better tissue specificitythan mRNAs. The inventors also found significant variability in lncRNAexpression among EC lines, consistent the observed heterogeneity amongECs. Given the central importance of ECs in vascular biology, thisdataset may provide a foundation to study the regulation and functionfor lncRNAs in various vascular development and disease models. Of note,the inventors also found many lncRNAs are highly expressed in ECs, butthose lncRNAs are not necessarily EC-specific (data not shown).

Those lncRNAs may also important function in cell types including ECs.Looking deep into the gene list, 91 lncRNAs of the 498 EC-enriched geneshave protein coding genes within 10-kb, and about a third of them showedparallel or inverse expression pattern to the associated genes.Functional enrichment analysis indicates that EC-enriched lncRNAs areassociated with genes involved in vascular development. Those lncRNAsmay be good candidates for further functional studies.

lncEGFL7OS is a Primate-Specific EC-Enriched lncRNA Required for ProperHuman Angiogenesis.

The inventors discovered lncEGFL7OS that is located in the oppositestrand neighboring the EGFL7/miR-126 gene. The expression of this lncRNAis enriched in ECs and highly vascularized tissues, which is consistentwith the expression of its host genes EGFL7 and miR-126. As to itsregulatory mechanisms, the inventors found that both lncEGFL7OS andmiR-126 were regulated by ETS1/2 factors in ECs through a bidirectionalpromoter. The inventors found that lncEGFL7OS is required for propervascular tube formation by using Matrigel assay in vitro and in vivo.Using a human choroid sprouting angiogenesis model the inventors newlydeveloped, the inventors further demonstrated that lncEGFL7OS isrequired for human sprouting angiogenesis. This study indicates thatthree different transcripts from the EGFL7/miR-126 locus, includinglncEGFL7OS, EGFL7 and miR-126, have important functions in angiogenesis.EGFL7 and miR-126 have been previously shown to regulate angiogenesis.EGFL7 is essential for vascular tube formation during vasculogenesis inzebrafish. Deletion of EGFL7 in mice resulted in several vasculardevelopmental defects and partial embryonic lethality, although thisphenotype was attributable by miR-126 loss-of-function by following upstudies. The importance of miR-126 in angiogenesis was demonstrated byloss-of-function studies in both mouse and zebrafish.

Targeted deletion of miR-126 in mice or miR-126 knockdown in zebrafishresulted in loss of vascular integrity and defective angiogenesis. It isintriguing that, in contrast to EGFL7 and miR-126, lncEGFL7OS representsa primate-specific mechanism in regulating angiogenesis, sincelncEGFL7OS only exists in human and potentially other primates. Newangiogenesis regulation mechanism through lncEGF7OS has evolved duringevolution underscores the importance and delicacy of EFGL7/miR-126 locusin angiogenesis. This study also highlights the importance of usinghuman (or primate) system to study the mechanism of angiogenesis.

Mechanism of lncEGFL7OS Action.

The inventors showed that the action of lncEGFL7OS reflects at leastpartially the regulation of expression of EGFL7 and miR-126. miR-126 hasbeen shown to promote MAP kinase and PI3K signaling in response to VEGFand FGF by targeting negative regulators of these signaling pathways,including Spred-1 and PIK3R2. Consistent with the downregulation ofmiR-126 by lncEGFL7OS silencing, the inventors found that thephosphorylation of ERK1/2 and AKT in response to VEGF is repressed bylncEGFL7OS silencing. Mechanistically, lncEGFL7OS likely acts anenhancer-like lncRNA that promotes EGFL7/miR-126 expression. The fullmechanism of lncEGF7 awaits further studies.

Therapeutic Implications.

Identifying angiogenic mechanisms that are conserved to human orhuman-specific is critical for developing therapeutics for humanvascular disorders. These studies have demonstrated that lncEGFL7OS is aprimate-specific lncRNA critical for human angiogenesis. This isdirectly translatable for human diseases involving abnormalangiogenesis. Particularly, AMD is the leading cause of blindness in theelderly, and choroidal neovascularization is a hallmark for wet AMD.Although anti-VEGF agents can markedly improve the clinical outcome ofwet AMD, they have been unable to induce complete angiogenesisregression, and only 30-40% of individuals experienced visionimprovement after treatment. The inventors developed a human choroidsprouting angiogenesis model and showed that lncEGFL7OS represses humanchoroid sprouting angiogenesis.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this disclosure havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the disclosure. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the disclosure as defined by theappended claims.

What is claimed is:
 1. A composition comprising an antagonist oflncEGFL7OS function in a pharmaceutically acceptable buffer, diluent ormedium, wherein said antagonist is one or more of si-LncEGFL7OS#1 andsi-LncEGFL7OS#2, or an expression vector coding for the same.
 2. Thecomposition of claim 1, further comprising a nucleic acid antagonist ofmiR-126.
 3. A method of inhibiting pathologic vascularization in asubject in need thereof comprising administering to the subject at riskof or suffering from pathologic vascularization an antagonistcomposition according to claim
 1. 4. The method of claim 3, wherein saidsubject is suffering from pathologic vascularization.
 5. The method ofclaim 3, wherein said subject is at risk of pathologic vascularization.6. The method of claim 3, wherein said antagonist is delivered to avasculature tissue, smooth muscle, ocular tissue, hematopoietic tissue,bone marrow, lung tissue or an epicardial tissue.
 7. The method of claim3, further comprising administering to said subject a secondaryanti-angiogenic therapy.
 8. The method of claim 3, wherein administeringcomprises systemic administration.
 9. The method of claim 3, whereinadministration is directly to or local to pathologic vascularization ora tissue at risk of pathologic vascularization.
 10. The method of claim3, wherein administering is by osmotic pump or catheter.
 11. Thecomposition of claim 1, wherein si-LncEGFL7OS#1 and/or si-LncEGFL7OS#2comprise at least one modified base.